Composition and method for treating cancer using herpes virus

ABSTRACT

The present invention provides a herpes virus in which a non-essential gene for replication is inactivated More particularly, the present invention provides a herpes virus in which a non-essential gene for replication present in a UL or US region is inactivated. More preferably, the non-essential gene for replication contains US3 or UL56. The herpes virus may be preferably a herpes simplex virus, and more preferably herpes simplex virus 1 or herpes simplex virus 2. The present invention provides a method, composition and use for treating various diseases or disorders including tumor and infectious diseases. The present invention also provides a method, composition and use for activating a prodrug.

TECHNICAL FIELD

The present invention relates to a novel herpes simplex virus(hereinafter may be herein abbreviated as HSV) construct. Morespecifically, the present invention relates to a method, use and acomposition for treatment, therapy, or prevention of various diseases ordisorders (e.g., cancer, bacterial infectious diseases, viral infectiousdiseases, and the like) using a novel herpes virus construct.

BACKGROUND ART

The first record of herpes virus in history dates back to the ancientGreece era before Christ. Herpes virus infrequently develops afterprimary infection, but hides in ganglia or the like for a long time.When the immune ability of a human is reduced, the virus is activatedand proliferates to develop symptoms. In this case, herpes virus targetsa wide range of tissues, such as skin, genital organs, eye ball, nerves,and the like. Even though antibodies which neutralize the virus arepresent in blood, symptoms often appear. It is thus believed that areduction in cellular immune function is involved in the onset ofsymptoms.

Examples of herpes virus transmissible to a human include herpes simplexvirus (HSV-1), herpes simplex virus (HSV-2), varicella zoster virus(VZV), EB virus (EBV), cytomegalovirus (HCMV), human herpes virus 6(HHV-6), human herpes virus 7 (HHV-7), human herpes virus 8 (HHV-8), andthe like.

HSV-1 causes gingivostomatitis or herpes facialis as a symptom ofprimary infection, and herpes labialis, herpes keratoconjunctivitis,herpes encephalitis as a symptom of recurrent infection. A major latentinfection site of HSV-1 is trigeminal ganglia. HSV-2 causes genitalherpes as a symptom of primary infection and genital herpes or neonatalherpes as a symptom of recurrent infection. A major latent infectionsite of HSV-2 is sacral ganglia. VZV causes chickenpox (varicella) as asymptom of primary infection and chickenpox and herpes zoster as asymptom of recurrent infection. A major latent infection site of VZV isdorsal root ganglion. EBV causes no symptom or causes infectiousmononucleosis as a symptom of primary infection and Burkitt's lymphoma,or rhinopharyngeal cancer as a symptom of recurrent infection. A majorlatent infection site of EBV is B cell. HCMV causes no symptom as asymptom of primary infection and causes pneumonia, cytomegalic inclusiondisease or the like as a symptom of recurrent infection. Latentinfection sites of HCMV are believed to be macrophage and bloodprogenitor cells. HHV-6 causes exanthema subitum as a symptom of primaryinfection and exanthema subitum (pneumonia) as a symptom of recurrentinfection. Latent infection sites of HHV-6 are believed to be macrophageand blood progenitor cells. HHV-7 causes exanthema subitum as a symptomof primary infection. A major latent infection site of HHV-7 is T cells.HHV-8 causes no symptom as a symptom of primary infection and causesKaposi's sarcoma, PEL, and Castleman's disease as a symptom of recurrentinfection. A major latent infection site of HHV-8 is B cells. Thus, avariety of infectious diseases are caused by herpes viruses.

An attenuated live vaccine against chickenpox virus is the onlysuccessful human herpes virus vaccine (Takahashi M., et al., Lancet,2:1288, 1974). However, this attenuated live virus was accidentallyobtained and is based on the characteristic that chickenpox is moreeffective to humoral immunity since it causes systemic infection due toviremia, unlike other herpes viruses. Therefore, a vaccine which can beuniversally utilized for herpes viruses is not yet available.

HSV is a representative herpes virus which is infectious to a human. HSVinfection results from inoculation of the virus to mucosa, or the virusinvading through a break in the skin. Most primary infections occur inthe neonatal periods, but most children with a primary infection quicklyimprove. HSV is transmitted to a baby from an infected nurser or moregenerally an infected mother (specifically, a baby encounters genitalherpes as the baby passes through the birth canal). In this case, HSVinfection causes serious symptoms. For example, disseminated neonatalherpes infection causes hepatitis or the like, so that a baby may die.

Once HSV is acquired in the body, HSV is held in the body over alifetime. During the latency period, HSV is localized in neurons insensory ganglia (in the case of facial lesions, trigeminal ganglia areusually involved), and an infected patient is a symptomatic. However,HSV is activated by stimuli, such as menstruation, excessive exposure tosunlight or cold wind, pituitary gland or adrenal gland hormones,allergic reactions, or fever. The activated HSV is replicated and takesover the mechanism of a host cell, producing infectious mature virusparticles and causing cell death. Symptoms caused by such a recurrentattack often appear on the mouth, face, and genital organs. For example,keratitis due to recurrent HSV is considered to be a major cause ofblindness. Further, HSV infection to genital organs is frequent, and theincidence of sexually transmitted diseases (e.g., genital herpes) causedby HSV is significantly high. More specifically, examples of recurrentHSV-induced diseases which are caused by the recurrent attack includemucocutaneous diseases, such as herpes labialis (a disorder on the lipsusually called “fever blister” or “cold sore”), gingivostomatitis (themouth and gingiva are covered with vesicles and the vesicles rupture toform ulcers), pharyngitis, tonsillitis, keratoconjunctivitis (keratitisor inflammation of the cornea, which progresses to dendriform ulcer andeventually to cicatrization of the cornea, resulting in blindness), andgenital herpes. In rare cases, HSV infection causes encephalitis, eczemaherpeticum, traumatic herpes, and hepatitis.

Herpes simplex virus 2 (hereinafter also referred to as HSV-2) inducesskin mucosa infection in genital organs. After infection, virus ismaintained in the sensory ganglia, and then activated to cause recurrentHSV infection (Price, R. W., Walz, M. A., Wohlenberg, C., and Notkins,A. L. (1975) “Latent infection of sensory ganglia with herpes simplexvirus: efficacy of immunization”, Science 188, 938-940). Manyresearchers have extensively studied immune reactions with HSV-2infection using animal models.

It has been reported that a major antigen-presenting cell (APC) is thedendritic cell (DC), including Langerhans cell (LC), and a number ofmacrophages (Mφ) and B cells in the vagina (Parr, M. B., and Parr, E. L.(1991), “Langerhans cells and T lymphocyte subsets in the murine vaginaand cervix”, Biol. Reprod. 44:491-498; Nandi, D., and Allison, J. P.(1993), “Characterization of neutrophiles and T lymphocytes associatedwith the murine vaginal epithelium”, Reg. Immunol., 5, 332-338).

It has been reported that T cells play an important role as a cytotoxicT lymphocyte (CTL), and produce antiviral cytokines against HSV-2infection (Milligan, G. N., and Bernstein, D. I. (1995), “Analysis ofherpes simplex virus-specific T cells in the murine female genital tractfollowing genital infection with herpes simplex virus type 2”, Virology212, 481-489.; Parr, M. B., and Parr, E. L. (1998), “Mucosal immunity toherpes simplex virus type 2 infection in the mouse vagina is impaired byin vivo depletion of T lymphocytes”, J. Virol. 72, 2677-2685; Milligan,G. N., Bernstein, D. I., and Bourne, N. (1998), “T lymphocytes arerequired for protection of the vaginal mucosae and sensory ganglia ofimmune mice against reinfection with herpes simplex virus type 2”, J.Immunol. 160, 6093-6100).

It has been reported that mutants lacking replication capability canelicit a wide spectrum of immune reactions (Morrison, L. A., Da Costa,X. J., and Knipe, D. M. (1998), “Influence of mucosal and parenteralimmunization with a replication-defective mutant of HSV-2 on immuneresponses and protection from genital infection”, Virology 243, 178-187:McLean, C. S., Ni Challanain, D., Duncan, I., Boursnell, M. E. G.,Jennings, R., and Inglis, S. C. (1996), “Induction of a protectiveimmune response by mucosal vaccination with a DISC HSV-1 vaccine”,Vaccine 14, 987-992).

It is known that infection with attenuated HSV-2 causes the flow ofHSV-2-specific T cell type 1 (TH1)-like CD4⁺ cells into the vagina(Milligan, G. N., Bernstein, D. I., and Bourne, N. (1998), “Tlymphocytes are required for protection of the vaginal mucosae andsensory ganglia of immune mice against reinfection with herpes simplexvirus type 2”, J. Immunol. 160, 6093-6100).

However, in the pathology of HSV diseases, a detailed relationshipbetween the role of a HSV-specific virus gene and immune reactions hasnot been fully clarified.

Thus, under the present circumstances, there is no decisive method forpreventing or treating diseases or disorders caused by herpes viruses.

As a cancer therapy, there are generally surgical excision,chemotherapy, radiation therapy, and the like at present. However, noneof these therapies have a sufficient effect on some types of cancers.For example, in the case of progressive pancreatic cancer andprogressive ovarian cancer, a favorable prognosis is not obtained.Particularly, when progressive pancreatic cancer or progressive ovariancancer is disseminated to peritonea, prognosis after surgical excisionis likely to fall short of expectations.

Therefore, an attempt is being made to develop gene therapy as a newmethod for treating cancer. The following gene therapies against cancerare being studied: (1) a method for inhibiting the growth of tumor cellsby controlling oncogenes using antisense or ribozyme, or by introducingantioncogenes; (2) a method for introducing a metabolically toxic gene(suicide gene) into tumor cells to cause them to commit suicide: (3) amethod for enhancing anti-tumor immunity by introducing genes; (4) amethod for protecting bone marrow stem cells using multidrug-resistantgenes for the purpose of improving the effect of chemotherapy; and thelike.

As a method of (2) as listed above, a method which employs the thymidinekinase of herpes simplex virus as a suicide gene is known. For example,the thymidine kinase of herpes simplex virus is introduced into cancercells, and thereafter, ganciclovir is administered. The ganciclovir isphosphorylated by the thymidine kinase of herpes simplex virus to beactivated. The activated ganciclovir inhibits the DNA polymerase ofcancer cells. Therefore, by introducing the thymidine kinase of herpessimplex virus into cancer cells, the growth of the cancer cells can besuppressed, and it is also possible that the cancer cells are completelydestroyed.

Since herpes simplex virus is a pathogenic virus, wild-type HSV cannotbe used for cancer therapy. Therefore, research is directed to the useof attenuated herpes simplex viruses for cancer therapy (WO96/39841).

Therefore, an attempt has been made to use gene therapy as a noveltherapy for cancer. As an example of such a gene therapy, ametabolically toxic gene (suicide gene) is introduced into tumor cellswhich are caused to commit suicide. More specifically, a method usingthe thymidine kinase of herpes simplex virus as a suicide gene is known.

As described above, herpes virus, such as herpes simplex virus (e.g.,herpes virus type 1 (HSV-1) capable of replication), may mediate thedestruction of tumor cells. Therefore, the use of a geneticallyengineered HSV-1 virus vector capable of replication is studied inassociation with antitumor therapy.

As such a HSV-1 virus vector, a γ34.5-deficient strain which lacks theγ34.5 gene (γ34.5) and therefore has a reduced level of neurotoxicityhas been used, so that a certain level of its antitumor effect wasdemonstrated. However, the γ34.5-deficient strain has not yet becomepractical.

The present inventors have concentrated on a gene encoding US2 of HSV-2(hereinafter abbreviated as US2 gene) and a gene encoding US3 of HSV-2(hereinafter abbreviated as US3 gene), and investigated the pathologicalroles of these genes using a US2-deficient mutant or a US3-deficient HSVgene recombinant. As a result, the present inventors revealed thatneither the US2 nor US3 gene is necessarily essential for thereplication of the respective viruses in cell culture, and theUS3-deficient HSV gene recombinant is significantly attenuated(Nishiyama, Y., Yamada, Y., Kurachi, R., and Daikoku, T (1992),“Construction of a US3 lacZ insertion Mutant of herpes simplex virustype 2 and characterization of its phenotype in vitro and in vivo”,Virology 190, 256-268; Daikoku, T., Yamashita, Y., Tsurumi, T., Maeno,K., and Nishiyama, Y (1993), “Purification and biologicalcharacterization of the protein kinase encoded by the US3 gene of herpessimplex virus type 2”, Virology 197, 685-694; Jiang, Y-H., Yamada, H.,Goshima, F., Daikoku T., Oshima, S., Wada, K., and Nishiyama, Y (1998),“Characterization of the herpes simplex virus type 2 (HSV-2) US2 geneproduct and a US2-deficient HSV-2 mutant”, J. Gen. Virol. 79,2777-2784).

Previous research on genital herpes using murine models revealed thatunlike infection with HSV-1 strain KOS, intravaginal infection withhighly virulent HSV-2 strain 186 fails to induce increases in activatedT cells within the vagina or a rapid increase of APC (antigen-presentingcell) in the early phase of infection (Inagaki-Ohara K, Daikoku T,Goshima F, Nishiyama Y, “Impaired induction of protective immunity byhighly virulent herpes simplex virus type 2 in a murine model of genitalherpes”, Arch Virol. 2000; 145(10): 1989-2002.).

Therefore, the objective of the present invention is to provide acharacteristic-modified herpes virus construct, the use thereof, or amethod using the same so as to treat or prevent diseases or disordersassociated with herpes virus, or other diseases or disorders.

DISCLOSURE OF THE INVENTION

The present invention provides a herpes virus in which a non-essentialgene for replication is inactivated. More particularly, the presentinvention provides a herpes virus in which a non-essential gene forreplication present in UL and US regions are inactivated. The presentinvention also provides a herpes virus in which a gene not involved inDNA and deoxyribonucleotide metabolism is inactivated. More preferably,the non-essential gene for replication contains US3 and UL56. The herpesvirus may be preferably a herpes simplex virus, and more preferablyherpes simplex virus 1 or herpes simplex virus 2. The present inventionprovides a method, composition and use for treating various diseases ordisorders including tumor and infectious diseases The present inventionalso provides a method, composition and use for activating a prodrug.

According to one aspect of the present invention, a herpes virus isprovided in which at least one non-essential gene for replicationthereof is inactivated.

In one embodiment of this invention, the non-essential gene forreplication is present in a UL region or a US region.

In one embodiment of this invention, the non-essential gene forreplication is selected from the group consisting of UL2, UL3, UL4, UL7,UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24, UL35, UL39, UL40,UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55, UL56, US1, US2,US3, US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication is a gene not involved in DNA and deoxyribonucleotidemetabolism.

In one embodiment of this invention, the gene not involved in DNA anddeoxyribonucleotide metabolism is selected from the group consisting ofUL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21, UL24, UL35,UL41, UL43, UL44, UL45, UL46, UL47, UL51, UL55, UL56, US1, US2, US3,US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication contains UL56 or US3.

In one embodiment of this invention, the non-essential gene forreplication contains UL39 and UL40.

In one embodiment of this invention, the non-essential gene forreplication contains UL39 and UL56.

In one embodiment of this invention, the non-essential gene forreplication contains UL2 and US3.

In one embodiment of this invention, the non-essential gene forreplication contains UL56.

In one embodiment of this invention, the non-essential gene forreplication contains US3.

In one embodiment of this invention, at least two non-essential genesfor replication are inactivated.

In one embodiment of this invention, the second non-essential gene forreplication and thereafter are selected from the group consisting ofRL1, RL2, ORFP, ORFO, RL3, UL2, UL3, UL4, UL7, UL10, UL11, UL12, UL13,UL16, UL20, UL20.5, UL21, UL24, UL39, UL40, UL41, UL43, UL43.5, UL44,UL45, UL46, UL47, UL56, UL51, UL55, UL56, US1, US1.5, US2, US3, US4,US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the herpes virus further comprisesan exogenous suicide gene.

In one embodiment of this invention, the herpes virus further comprisesa carboxyesterase gene.

In one embodiment of this invention, the virus has ability to selectcancer cells.

In one embodiment of this invention, the inactivation includes at leastone nucleotide substitution, addition, deletion or modification in thesequence of the non-essential gene for replication.

In one embodiment of this invention, the virus is a modified herpessimplex virus.

In one embodiment of this invention, the virus is a modified HSV-1 orHSV-2.

According to another embodiment of the present invention, apharmaceutical composition comprises a herpes virus wherein at least onenon-essential gene for replication thereof is inactivated, and apharmaceutically acceptable carrier.

In one embodiment of this invention, the non-essential gene forreplication is present in a UL region or a US region.

In one embodiment of this invention, the non-essential gene forreplication is selected from the group consisting of UL2, UL3, UL4, UL7,UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24, UL35, UL39, UL40,UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55, US1, US2, US4,US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication is a gene not involved in DNA and deoxyribonucleotidemetabolism.

In one embodiment of this invention, the gene not involved in DNA anddeoxyribonucleotide metabolism is selected from the group consisting ofUL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21, UL24, UL35,UL41, UL43, UL44, UL45, UL46, UL47, UL51, UL55, UL56, US1, US2, US3,US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication contains UL56 or US3.

In one embodiment of this invention, the inactivation includes at leastone nucleotide substitution, addition, deletion or modification in thesequence of the non-essential gene for replication.

In one embodiment of this invention, a signal for stopping translationis inserted into the sequence of the non-essential gene for replication.

In one embodiment of this invention, the signal for stopping translationis a polyadenylation signal.

In one embodiment of this invention, the virus is a modified herpessimplex virus.

In one embodiment of this invention, the virus is a modified HSV-1 orHSV-2.

In one embodiment of this invention, the pharmaceutical compositionfurther comprises a prodrug capable of converting the attenuated herpesvirus into an active form.

In one embodiment of this invention, the prodrug is selected from thegroup consisting of ganciclovir, acyclovir, taxol, and camptothecin.

In one embodiment of this invention, the composition is used fortreatment of tumor.

In one embodiment of this invention, the pharmaceutical compositionfurther comprises at least one drug for treatment of tumor.

In one embodiment of this invention, the composition is used to enhancean anticancer action of the prodrug which is converted into an activeform by the herpes virus.

In one embodiment of this invention, the composition is used fortreatment of an infectious disease.

In one embodiment of this invention, the infectious disease is caused bya herpes virus.

In one embodiment of this invention, the pharmaceutical compositioncomprises a gene derived from a pathogen of the infectious disease.

In one embodiment of this invention, the pharmaceutical compositioncomprises L1BR1.

In one embodiment of this invention, the composition is in the form of avaccine.

In one embodiment of this invention, the composition is an agent fortreating or preventing a disease or disorder caused by infection of aherpes virus.

In one embodiment of this invention, the disease caused by the infectionof a herpes virus is a sexually transmitted disease.

In one embodiment of this invention, the composition is used fortreatment or prevention of an infectious disease.

In one embodiment of this invention, the pharmaceutical compositionfurther comprises a pathogen of the infectious disease.

In one embodiment of this invention, the pathogen of the infectiousdisease is a virus or bacterium.

In one embodiment of this invention, the pathogen of the infectiousdisease is selected from the group consisting of HIV, influenza virus,and rotavirus.

In one embodiment of this invention, a method for treating or preventingan infectious disease or tumor, comprises the step of administering apharmaceutical composition comprising a herpes virus, at least onenon-essential gene for replication thereof being inactivated, and apharmaceutically acceptable carrier, to a subject requiring thetreatment or prevention.

In one embodiment of this invention, the non-essential gene forreplication is present in a UL region or a US region.

In one embodiment of this invention, the non-essential gene forreplication is selected from the group consisting of UL2, UL3, UL4, UL7,UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24, UL35, UL39, UL40,UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55, UL56, US1, US2,US3, US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication is a gene not involved in DNA and deoxyribonucleotidemetabolism.

In one embodiment of this invention, the gene not involved in DNA anddeoxyribonucleotide metabolism is selected from the group consisting ofUL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21, UL24, UL35,UL41, UL43, UL44, UL45, UL46, UL47, UL51, UL55, UL56, US1, US2, US3,US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication contains US3 or UL56.

In one embodiment of this invention, the inactivation includes at leastone nucleotide substitution, addition, deletion or modification.

In one embodiment of this invention, the herpes virus is a modifiedherpes simplex virus.

In one embodiment of this invention, the herpes virus is a modifiedHSV-1 or HSV-2.

In one embodiment of this invention, the method further comprises thestep of administering a prodrug which is converted into an active formby a herpes virus.

In one embodiment of this invention, the prodrug is selected from thegroup consisting of ganciclovir, acyclovir, taxol, and camptothecin.

In one embodiment of this invention, the method comprises the step ofadministering at least one drug for treatment of tumor.

In one embodiment of this invention, the method further comprises thestep of administering at least one drug for treatment of an infectiousdisease.

In one embodiment of this invention, the tumor is selected from thegroup consisting of ovarian cancer, liver cancer, pancreatic cancer,bladder cancer, urethra cancer, large intestine cancer, skin cancer,malignant melanoma, osteosarcoma, head and neck squamous cell carcinoma,and stomach cancer.

According to another aspect of the present invention, a method isprovided for enhancing an anticancer action of a prodrug which isconverted into an active form by a herpes virus. The method comprisesthe step of administering a pharmaceutical composition comprising aherpes virus, at least one non-essential gene for replication thereofbeing inactivated, and a pharmaceutically acceptable carrier to asubject requiring the enhancement of an anticancer action.

In one embodiment of this invention, the non-essential gene forreplication is present in a UL region or a US region.

In one embodiment of this invention, the non-essential gene forreplication is selected from the group consisting of UL2, UL3, UL4, UL7,UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24, UL35, UL39, UL40,UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55, UL56, US1, US2,US3, US4, US5, US7, US8, US8.5, US9, US10, US11, and US12.

In one embodiment of this invention, the non-essential gene forreplication is a gene not involved in DNA and deoxyribonucleotidemetabolism.

In one embodiment of this invention, the gene not involved in DNA anddeoxyribonucleotide metabolism is selected from the group consisting ofUL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21, UL24, UL35,UL41, UL43, UL44, UL45, UL46, UL47, ULSI, UL55, UL56, US1, US2, US3,US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication contains US3 or UL56.

In one embodiment of this invention, the prodrug is selected from thegroup consisting of ganciclovir, acyclovir, taxol, and camptothecin.

In one embodiment of this invention, the herpes virus is a modifiedherpes simplex virus.

In one embodiment of this invention, the herpes virus is a modifiedHSV-1 or HSV-2.

According to another aspect of the present invention, a method forreducing the severity or infection rate of a herpes virus, comprises thestep of administering a vaccine comprising a herpes virus, at least onenon-essential gene for replication thereof is inactivated, to a subjectrequiring the treatment or prevention.

In one embodiment of this invention, the non-essential gene forreplication is present in a UL region or a US region.

In one embodiment of this invention, the non-essential gene forreplication is selected from the group consisting of UL2, UL3, UL4, UL7,UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24, UL35, UL39, UL40,UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55, UL56, US1, US2,US3, US4, US5, US7, US8, US8.5, US9, US10, US11, and US12.

In one embodiment of this invention, the non-essential gene forreplication is a gene not involved in DNA and deoxyribonucleotidemetabolism.

In one embodiment of this invention, the gene not involved in DNA anddeoxyribonucleotide metabolism is selected from the group consisting ofUL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21, UL24, UL35,UL41, UL43, UL44, UL45, UL46, UL47, UL51, UL55, UL56, US1, US2, US3,US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication contains US3 or UL56.

In one embodiment of this invention, the herpes virus is a modifiedherpes simplex virus.

In one embodiment of this invention, the herpes virus is a modifiedHSV-1 or HSV-2.

According to another aspect of the present invention, use of a herpesvirus for producing a pharmaceutical composition for treating orpreventing tumor is provided. At least one non-essential gene forreplication of the herpes virus is inactivated.

In one embodiment of this invention, the non-essential gene forreplication is present in a UL region or a US region.

In one embodiment of this invention, the non-essential gene forreplication is selected from the group consisting of UL2, UL3, UL4, UL7,UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24, UL35, UL39, UL40,UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55, UL56, US1, US2,US3, US4, US5, US7, US8, US8.5, US9, US10, US11, and US12.

In one embodiment of this invention, the non-essential gene forreplication is a gene not involved in DNA and deoxyribonucleotidemetabolism.

In one embodiment of this invention, the gene not involved in DNA anddeoxyribonucleotide metabolism is selected from the group consisting ofUL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21, UL24, UL35,UL41, UL43, UL44, UL45, UL46, UL47, UL51, UL55, UL56, US1, US2, US3,US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication contains US3 or UL56.

In one embodiment of this invention, the herpes virus is a modifiedherpes simplex virus.

In one embodiment of this invention, the herpes virus is a modifiedHSV-1 or HSV-2.

According to another aspect of the present invention, use of a herpesvirus for producing a pharmaceutical composition for enhancing ananticancer action of a prodrug which is converted into an active form bythe herpes virus, is provided. At least one non-essential gene forreplication of the herpes virus is inactivated.

In one embodiment of this invention, the non-essential gene forreplication is present in a UL region or a US region.

In one embodiment of this invention, the non-essential gene forreplication is selected from the group consisting of UL2, UL3, UL4, UL7,UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24, UL35, UL39, UL40,UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55, UL56, US1, US2,US3, US4, US5, US7, US8, US8.5, US9, US10, US11, and US12.

In one embodiment of this invention, the non-essential gene forreplication is a gene not involved in DNA and deoxyribonucleotidemetabolism.

In one embodiment of this invention, the gene not involved in DNA anddeoxyribonucleotide metabolism is selected from the group consisting ofUL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21, UL24, UL35,UL41, UL43, UL44, UL45, UL46, UL47, ULSI, UL55, UL56, US1, US2, US3,US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication contains US3 or UL56.

In one embodiment of this invention, the herpes virus is a modifiedherpes simplex virus.

In one embodiment of this invention, the herpes virus is a modifiedHSV-1 or HSV-2.

According to another aspect of the present invention, use of a herpesvirus for producing a pharmaceutical composition for reducing theseverity or infection rate of a herpes virus, is provided. At least onenon-essential gene for replication of the herpes virus is inactivated.

In one embodiment of this invention, the non-essential gene forreplication is present in a UL region or a US region.

In one embodiment of this invention, the non-essential gene forreplication is selected from the group consisting of UL2, UL3, UL4, UL7,UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24, UL35, UL39, UL40,UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55, UL56, US1, US2,US3, US4, US5, US7, US8, US8.5, US9, US10, US11, and US12.

In one embodiment of this invention, the non-essential gene forreplication is a gene not involved in DNA and deoxyribonucleotidemetabolism.

In one embodiment of this invention, the gene not involved in DNA anddeoxyribonucleotide metabolism is selected from the group consisting ofUL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21, UL24, UL35,UL41, UL43, UL44, UL45, UL46, UL47, UL51, UL55, UL56, US1, US2, US3,US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

In one embodiment of this invention, the non-essential gene forreplication contains US3 or UL56.

In one embodiment of this invention, the pharmaceutical composition is avaccine.

In one embodiment of this invention, the herpes virus is a modifiedherpes simplex virus.

In one embodiment of this invention, the herpes virus is a modifiedHSV-1 or HSV-2.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a graph showing a cancer treating effect obtained byinoculating an attenuated HSV according to the present invention.

FIG. 2 is a graph showing comparison of a cancer treating effect betweenthe attenuated HSV of the present invention and known anticancer agents.

FIG. 3 shows clinical features of BALB/c mice which were intravaginallywere infected with 2×10⁴ PFU of wild type 186, wild type YY2, or L1BR1after E/DP treatment. Photographs were taken on day 7 after intravaginalinfection.

FIG. 4 shows the survival rates of mice which were intravaginallyinfected with 2×10⁴ PFU of wild type 186, wild type YY2, or L1BR1. Ineach case, the number of tests was n=7. It should be noted that in thisfigure, filled circles indicate wild type 186, unfilled squares indicatewild type YY2, and unfilled circles indicate L1BR1.

FIG. 5 shows viral clearance from the vaginas of mice infected with wildtype 186, wild type YY2 or L1BR1. Vaginal wash on day 0 was collectedtwo hours after intravaginal infection of 2×10⁴ PFU of wild type 186,wild type YY2 or L1BR1. Values in three separate experiments arerepresented by ±standard deviation (SD). It should be noted that in thisfigure, filled circles indicate wild type 186, unfilled squares indicatewild type YY2, and unfilled circles indicate L1BR1. Also, in the figure,** indicates that a significant difference is p<0.001 when compared withvalues for mice infected with wild type 186. In the figure, * indicatesthat the significant difference is p<0.01.

FIGS. 6A through 6F show the vaginal mucosae of BALB/c mice used inhistochemical study in examples. FIGS. 6A and 6D show the vaginalmucosae of mock-infected mice. FIGS. 6B and 6E show the vaginal mucosaeof mice infected with wild type 186. FIGS. 6C and 6F show the vaginalmucosae of mice infected with L1BR1. G, H and I indicate histologicalchanges in the vaginal walls of mice infected with wild type 186 (G),wild type YY2 (H) and L1BR1 (I) on day 5 after inoculation. It should benoted that A to I are microscopic photographs at a magnification of 100.A, B, C, G, H and I show heamatoxylin-eosin stain. D, E and F showdetection of HSV antigens.

FIG. 7A shows the number of vaginal MNCs infected with wild type 186,wild type YY2 or L1BR1. The number of vaginal MNCs was measured bycounting surviving cells stained with trypan blue, which were collectedfrom mice infected with each virus. It should be noted that in thisfigure, filled circles indicate wild type 186, unfilled squares indicatewild type YY2, and unfilled circles indicate L1BR1.

FIG. 7B shows the kinetics of Fas expressed in vaginal EC. Vaginal ECisolated from mice infected with wild type 186, wild type YY2, or L1BR1was stained with anti Fas mAb.

FIG. 8 shows vaginal MNCs isolated from HIV-infected mice, which werestained with CD11b and CD40 for detection of APC, and with labeled mAbfor detection of T cell. Values in three separate experiments arerepresented by ±standard deviation (SD). In the figure, ** indicatesthat a significant difference was p<0.001 when compared with values formice infected with wild type 186. In the figure, * indicates that thesignificant difference was p<0.01.

FIG. 9 shows the amount of cytokines generated in vaginal wash. Thevaginal wash was collected from mice infected with wild type 186, wildtype YY2, or L1BR1 on day 0, 2, and 4. The levels of IL-12, IFN-γ andIL-4 were measured by ELISA. Values in three separate experiments arerepresented by ±standard deviation (SD). In this figure, ** indicatesthat a significant difference is p<0.05 when compared with values formice infected with wild type 186. In the figure, * indicates that thesignificant difference is p<0.01.

FIG. 10A is a schematic diagram showing a gene of HSV-2. This schematicdiagram is accompanied by a map unit.

FIG. 10B is a cleavage map of HVS-2 strain 186 by Hind III.

FIG. 10C shows the corresponding positions of restriction enzyme(restriction endonuclease cleavage site) on the map, which relate to thepresent invention. It should be noted that in the figure, H indicatesrestriction enzyme sites of Hind III, B indicates restriction enzymesites of Bam HI, G indates restriction enzyme sites of Bal II, Xindicates restriction enzyme sites of Xba I.

FIG. 10D indicates the position of the US3 gene in a 4.8-kb Hind III-XbaI fragment precloned by pLHX which is pBluescript.

FIG. 10E shows a 4.3-kb Bam HI fragment containing a gene encoding lazZfused with a HSV-1 β8 promoter and a polyadenylation signal derived fromSV40 at 5′ end and 3′ end, respectively. The 4.3-kb Bam HI fragment wasinserted into restriction enzyme sites of pLHX by Bal II, therebypreparing a gene recombinant pHZL1.

FIG. 11 is a diagram showing comparison of survival time betweenUL56-deficient HSV (strain HF; MNO10) inoculation and UL39-inactivatedHSV inoculation. In the figure, unfilled diamonds indicate a control,filled squares indicate mice given the UL39-inactivated HSV at one time,unfilled triangles indicate mice given the UL39-inactivated HSV at twotimes, and crisscrosses indicate mice given strain RF at one time.

FIG. 12 is a diagram showing the survival time when UL56-deficient HSV(MNO10) were consecutively administered. In the figure, filled diamondsindicate a control, and unfilled triangles indicate mice consecutivelygiven strain HF. The body weight change thereof is also shown.

FIG. 13A is a diagram showing the structure of a genome used in Example9. L1BR1 is HSV-2 in which the US3 gene thereof is inactivated. HF isHSV-1 in which the UL56 gene thereof is inactivated. HL is a hybrid ofL1BR1 and HF. A L region is HSV-1 while-a S region is HSV-2. Both UL56and US3 are deficient. The genome structure is confirmed by the PCRanalysis below.

FIG. 13B is a diagram showing the structure of a genome used in Example13. HF is HSV-1 in which the UL56 gene thereof is inactivated. Hh isHSV-1 in which the UL39 and UL56 genes thereof are inactivated.UL39-inactivated HSV is HSV-1 in which the UL39 gene is inactivated. Alist of mutated bases is also shown.

FIG. 13C is a diagram for explaining inactivation of HF in detail. FIG.13D shows a gene sequence in the vicinity of the UL56 gene insertedbetween TRL and the UL region. FIG. 13E shows a gene sequence in thevicinity of the UL52 gene inserted between TRL and the UL region. FIG.13F shows an inherent gene sequence in the vicinity of the UL56 gene.

FIG. 14 is a graph showing the antitumor effect of HSV-2 in which theUS3 gene thereof is inactivated.

FIG. 15 is a graph showing the antitumor effect of HSV-2 in which theUL56 gene thereof is inactivated.

BEST MODE FOR CARRYING OUT THE-INVENTION

It should be understood throughout the present specification thatarticles for a singular form (e.g., “a”, “an”, “the”, etc. in English;“ein”, “der”, “das”, “die”, etc. and their inflections in German; “un”,“une”, “le”, “la”, etc. in French; “un”, “una”, “el”, “la”, etc. inSpanish, and articles, adjectives, etc. in other languages) include theconcept of their plurality unless otherwise mentioned. It should be alsounderstood that the terms as used herein have definitions typically usedin the art unless otherwise mentioned. Documents, patents or patentapplications cited herein are herein incorporated by reference in theirentirety.

The term “herpes virus” as used herein refers to any viruses belongingto the family herpes virus. A herpes virus comprises about 162capsomeres which include double helix DNA having a molecular weight ofabout 80-150×10⁶ Da around a core protein. A herpes virus ischaracterized by latent infection. A herpes virus particle is in theshape of a sphere having a diameter of about 100-200 nm and has aregular icosahedron-like capsid of a diameter of around about 100 nminside its envelope. The genome thereof is double-stranded DNA. The sizeof the genome is 152260 base pairs for herpes simplex virus type1,172282 base pairs for EBVirus, 229400 base pairs for cytomegalovirus,for example. The virus is multiplied within a cell nucleus to forminclusion bodies which are positive to Feulgen reaction. The virus ismultiplied well in a natural host, epithelial cells of an experimentalanimal, such as skin and mucosa, central nerve tissue, and the like. Thefamily herpes virus is divided into three: the subfamily alpha herpesvirus (e.g., human herpes virus 1 (herpes simplex virus type 1; HSV-1),human herpes virus 2 (herpes simplex virus type 2: HSV-2), Allertonvirus, B virus, bovine mammilitis virus (BMV), feline rhinotracheitisvirus, infectious laryngotracheitis virus ILT virus, varicella zostervirus (VZV), and the like); the subfamily beta herpes virus (e.g.,cytomegalovirus, human herpes virus 6 (HHV-6), human herpes virus 7(HHV-7), and the like); the subfamily gamma herpes virus (e.g., humanherpes virus 4 (HHV-4), human herpes virus 8 (HHV-8), EBVirus (EBV),Marek's disease virus, and the like). Preferably, the herpes of theinvention derived of the subfamily alpha herpes virus.

The term “herpes simplex virus” or “HSV” as used herein refers to any ofviruses of the genus simplex virus of the subfamily alpha herpes virusof the family herpes virus. Herpes simplex virus is 100 to 200 mm indiameter and has an envelope containing a viral glycoprotein at itsoutermost layer, in which an icosahedral capsid is included. A constructcalled tegument is present between the capsid and the envelope, in whicha number of viral proteins are contained. In the central portion(core)of the capsid, there is double-stranded DNA (about 150,000 basepairs). At least 74 genes are present in the genomic DNA, about half ofwhich are non-essential genes for reproduction of cultured cells(accessory genes). Reproduction of genomic DNA and formation of thecapsid are carried out within the nucleus. The capsid including DNA istransferred from the nucleus to the cytoplasm by budding from nuclearmembrane. HSV includes two virus species called, representatively, type1 (HSV-1) and type 2 (HSV-2), which are closely related to each other. Amutant of HSV-1 and HSV-2 is employed in the present invention. In somecases, HSV-2 is preferable. The term “attenuated HSV” as used hereinrefers to HSV whose toxicity is reduced by modification or the like.

The terms “non-essential gene for replication” and “accessory gene” areherein used interchangeably. Even if such a gene is absent in a virus,the growth of the virus is maintained. Such a gene is located in a ULregion or US region and constitutes a unique gene in the genomic DNA.Preferable examples of such a gene include, but are not limited to, UL2,UL3, UL4, UL7, UL10, UL11, UL12, UL13, UL14, UL16, UL20, UL21, UL24,UL35, UL39, UL40, UL41, UL43, UL44, UL45, UL46, UL47, UL50, UL51, UL55,UL56, US1, US2, US3, US4, US5, US7, US8, US8.5, US9, US10, US11, US12,and the like for herpes simplex virus such as HSV-1 and HSV-2. For otherherpes viruses, genes corresponding to the above-described genes may beinactivated.

In one embodiment, when a plurality of non-essential genes forreplication are inactivated, the second gene and thereafter may be, butis not limited to, RL1, RL2, ORFP, ORFO, RL3, UL2, UL3, UL4, UL7, UL10,UL11, UL12, UL13, UL16, UL20, UL20.5, UL21, UL24, UL39, UL40, UL41,UL43, UL43.5, UL44, UL45, UL46, UL47, UL50, UL51, UL55, UL56, US1,US1.5, US2, US3, US4, US5, US7, US8, US8.5, US9, US10, US11, US12, andthe like.

In a preferred embodiment, an example of the inactivated gene in thepresent invention is a gene which is not involved in DNA anddeoxyribonucleotide metabolism. Although the present invention is notintended to be constrained by theory, inactivation of genes not involvedin DNA and deoxyribonucleotide metabolism can dramatically increasesafety and/or specificity to humans. Such genes not involved in DNA anddeoxyribonucleotide metabolism, exclude the genes UL2, UL12, UL23, UL39,UL40 and UL50. Preferably, such genes not involved in DNA anddeoxyribonucleotide metabolism lie in the US or UL region. Thus, genesnot involved in DNA and deoxyribonucleotide metabolism include but arenot limited to UL3, UL4, UL7, UL10, UL11, UL13, UL14, UL16, UL20, UL21,UL24, UL35, UL41, UL43, UL44, UL45, UL46, UL47, UL51, UL55, UL56, US1,US2, US3, US4, US5, US7, US8, US8.5, US9, US10, US11 and US12.

The attenuated HSV of the present invention can be multiplied favorablyin cancer cells. Preferably, such a HSV has the ability to select acancer cell.

An example of an attenuated HSV with at least one accessory geneinactivated is one whose UL39 or UL40 is inactivated. In this case, anattenuated HSV, in which UL39 or UL40 is inactivated along with at leastone other accessory gene, is preferable.

The attenuated HSV in which UL39 or UL40 is inactivated must rely on ahost for ribonucleotide reductase which is encoded by the inactivatedgene, as it is required for DNA and deoxyribonucleotide metabolism.Therefore, when the attenuated HSV in which UL39 or UL40 is inactivatedis inoculated into a cancer patient, the HSV can be multiplied favorablyin vigorously dividing cancer cells. The cancer cells infected with suchan attenuated HSV are suppressed from being multiplied due to thecytopathic effect of various proteins of the virus, potentially leadingto their death.

When in addition to UL39 or UL40, at least one other accessory gene isinactivated, the virus can be multiplied favorably in cancer cells atsubstantially the same rate while the pathogenicity thereof to the hostcan be further reduced.

Examples of the attenuated HSV in which UL39 or UL40 and at least oneother accessory gene are inactivated include an attenuated HSV in whichUL39 (or UL40), UL55 and UL56 are inactivated, an attenuated HSV inwhich UL39 (or UL40) and UL2 are inactivated, an attenuated HSV in whichUL39 (or UL40) and US3 are inactivated, an attenuated HSV in which UL39(or UL40), UL2 and US3 are inactivated, and the like.

The attenuated HSV in which UL39 (or UL40), UL55 and UL56 areinactivated and the attenuated HSV in which UL39 (or UL40) and UL2 areinactivated have a higher level of safety than that of the attenuatedHSV in which only UL39 (or UL40) is inactivated. The attenuated HSV inwhich UL39 (or UL40) and US3 are inactivated and the attenuated HSV inwhich UL39 (or UL40), UL2 and US3 are inactivated can cause apoptosis ina cancer cell as well as having a high level of ability to select cancercells.

In another embodiment, a HSV in which US3 and/or UL56 is inactivated isprovided.

The term “a gene is inactivated” as used herein indicates that at leastone function of the gene is impaired, or preferably substantiallyeliminated. The method for inactivation is not particularly limited.Examples of such methods include the following known methods.

For example, a non-essential HSV gene for replication (e.g., US3 gene)is treated so as not to be translated (e.g., a promoter is altered so asnot to be expressed); or a particular non-essential gene for replicationout of the HSV genes (e.g., US3 gene) is subjected to recombination(e.g., insertion of another sequence, removal of a partial sequence,modification of bases, substitution of bases, and the like), so that therecombined gene is translated into a protein different from US3 (e.g.,insertion of another sequence, removal of a partial sequence,modification of amino acids, substitution of amino acids, and the like).

A gene recombination process for preparing the above-described generecombinants is not particularly limited. For example, a part orentirety of a particular non-essential gene for replication (e.g., US3gene) is deleted; a part or entirety of a particular non-essential genefor replication is substituted; a part of a particular non-essentialgene for replication is inverted, a part of the US3 gene is repeated; apart of a particular non-essential gene for replication is translocated;a gene fragment is inserted in a particular non-essential gene forreplication so as to interrupt the particular non-essential gene forreplication; and the like. Among them, deleting a part or entirety of aparticular non-essential gene for replication, substituting a part orentirety of particular non-essential gene for replication, or insertinga gene fragment in a particular non-essential gene for replication so asto interrupt the particular non-essential gene for replication ispreferable. Particularly, inserting a gene fragment in a particularnon-essential gene for replication so as to interrupt the particularnon-essential gene for replication is more preferable. Even morepreferably, the gene fragment to be inserted contains a signal whichstops translation. Among signals which stop translation, apolyadenylation signal derived from SV40 is preferable.

The term “protein”, “polypeptide” and “peptide” are herein usedinterchangeably and refer to a polymer consisting of a series of aminoacids. The term “amino acid” refers to an organic molecule containing acarbon atom(s) with a carboxy group(s) and an amino group(s). An aminoacid is herein preferably any of twenty naturally occurring amino acidsbut not limited to them.

A certain amino acid may be substituted with another amino acid withoutclear reduction or loss of interactive binding capability, for example,in a protein structure, such as a cationic region or a binding site fora substrate molecule. The biological function of a certain protein isdetermined by the interaction capability and properties of the protein.Therefore, even if substitution of a particular amino acid is performedin an amino acid sequence (or at the DNA code sequence level), a proteinmay maintain its original properties after the substitution. Therefore,peptides disclosed herein or DNA encoding the peptides may be modifiedin various manners without clearly impairing their biological utility.

When the above-described modifications are designed, the hydrophobicityindexes of amino acids may be taken into consideration. The hydrophobicamino acid indexes play an important role in providing a protein with aninteractive biological function, which is generally recognized in theart (Kyte. J and Doolittle, R. F., J. Mol. Biol. 157(1):105-132, 1982).The hydrophobic property of an amino acid contributes to the secondarystructure of a generated protein and then regulates interactions betweenthe protein and other molecules (e.g., enzymes, substrates, receptors,DNA, antibodies, antigens, etc.). Each amino acid is given ahydrophobicity index based on the hydrophobicity and charge propertiesthereof as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamic acid (−3.5); glutamine (−3.5); aspartic acid(−3.5): asparagine(−3.5); lysine (−3.9); and arginine (−4.5)).

It is well known that if a certain amino acid is substituted withanother amino acid having a similar hydrophobicity index, a resultantprotein may still have a biological function similar to that of theoriginal protein (e.g., a protein having an equivalent enzymaticactivity). For such an amino acid substitution, the hydrophobicity indexis preferably within ±2, more preferably within ±1, and even morepreferably within ±0.5. It is understood in the art that such an aminoacid substitution based on the hydrophobicity is efficient. As describedin U.S. Pat. No. 4,554,101, amino acid residues are given the followinghydrophilicity indexes: arginine (+3.0); lysine (+3.0); aspartic acid(+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2);glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1);alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); and tryptophan (−3.4). It is understood that anamino acid may be substituted with another amino acid which has asimilar hydrophilicity index and can still provide a biologicalequivalent. For such an amino acid substitution, the hydrophilicityindex is preferably within ±2, more preferably ±1, and even morepreferably ±0.5.

The term “conservative substitution” as used herein refers to amino acidsubstitution in which a substituted amino acid and a substituting aminoacid have similar hydrophilicity indexes or/and hydrophobicity indexes.Examples of the conservative substitution include, but are not limitedto, substitutions within each of the following groups: arginine andlysine; glutamic acid and aspartic acid; serine and threonine; glutamineand asparagine; and valine, leucine, and isoleucine, which are wellknown to those skilled in the art.

The term “nonconservative substitution” as used herein refers tosubstitution which is not a conservative substitution as described aboveand by which a function of a protein is altered. Examples ofnonconservative substitution include, but are not limited to,substitution between each of the following groups: arginine and lysine;glutamic acid and aspartic acid; serine and threonine; glutamine andasparagine: valine, leucine, and isoleucine; and the like, which arewell known to those skilled in the art.

To prepare functionally equivalent polypeptides, amino acid addition,deletion or modification can be performed other than amino acidsubstitution. Amino acid substitution means to substitute at least oneamino acid (e.g., 1 to 10 amino acids, preferably 1 to 5 amino acids,and more preferably 1 to 3 amino acids) in an original peptide with thesame number of other amino acids. Amino acid addition means to add atleast one amino acid (e.g., 1 to 10 amino acids, preferably 1 to 5 aminoacids, and more preferably 1 to 3 amino acids) to an original peptidechain. Amino acid deletion means to delete at least one amino acid(e.g., 1 to 10 amino acids, preferably 1 to 5 amino acids, and morepreferably 1 to 3 amino acids) from an original peptide. Amino acidmodification includes, but is not limited to, amidation, carboxylation,sulfation, halogenation, alkylation, glycosylation, phosphorylation,hydroxylation, acylation (e.g., acetylation), and the like. Asubstituted or added amino acid may be a naturally occurring amino acid,a non-naturally occurring amino acid, or an amino acid analog. Anaturally occurring amino acid is preferable.

A nucleic acid molecule encoding the polypeptide of the presentinvention used herein may have base deletion (s) (a part of the nucleicacid sequence is deleted), substitution(s) (a part of the nucleic acidsequence is substituted with other bases), or addition(s) (a part ofanother nucleic acid sequence is added) as long as the expressedpolypeptide has substantially the same activity as the polypeptide ofthe present invention, if the expressed peptide is intended to exhibitthe same or similar function. Alternatively, another nucleic acid may beligated to 5′ end and/or 3′ end. Further, a nucleic acid molecule whichhybridizes to a gene encoding the polypeptide of the present inventionunder stringent conditions and which encodes a polypeptide havingsubstantially the same function as that of the polypeptide of thepresent invention. A method for preparing such a nucleic acid moleculeis known in the art and is used in the present invention.

A part of the nucleic acid sequence of the polypeptide of the presentinvention may be subjected to deletion or substitution, or a part ofanother nucleic acid sequence may be added to the nucleic acid sequenceof the polypeptide so that the activity of the expressed polypeptide issubstantially different from that of the original polypeptide orpreferably eliminated, if the present invention is intended to modify,or preferably inactivate, a function of a polypeptide. Alternatively,another nucleic acid may be ligated to the 5′ end and/or 3′ end.

The term “expressibly incorporated” as used herein indicates that asuicide gene of interest is incorporated downstream of a promotersequence described below, for example.

The attenuated HSV of the present invention can be obtained by insertingthe above-described exogenous suicide gene downstream of a promotersequence using a HSV vector into which the promoter sequence and aterminator sequence are incorporated so as to inactivate an accessorygene, for example.

A promoter is not particularly limited if the promoter functions as apromoter in tumor cells. A tumor-specific promoter and a HSV-derivedpromoter are preferable. Examples of such a promoter, which is alsouseful in practicing the present invention (particularly, in productionof a gene vaccine for humans), include, but are not limited to,promoters derived from simian virus 40 (SV40 ), murine mammary glandtumor virus (MMTV), human immunodeficiency virus (HIV) (HIV longterminal repeat sequence (LTR) promoter), Moloney's virus, ALV,cytomegalovirus (CMV) (CMV immediate early promoter), Epstein-Barr virus(EBV), Rous sarcoma virus (RSV), and promoters from human genes, suchas, human actin, human myosin, human hemoglobin, human muscularcreatine, and human metallothionein.

A tumor cell-specific promoter includes a promoter which is inducedselectively or at a high level in a particular type of cell or tumorcell, such as a cancer embryonal protein promoter (e.g., CEA, AFP, andthe like), atyrosinase promoter, an albumin promoter, a stress-inducedGRP78/Bip promoter, and the like.

Examples of a HSV-derived promoter include a HSV primary protein UL29promoter, a HSV UL39 promoter, and the like.

Examples of a polyadenylation signal useful for practice of the presentinvention (particularly, in production of a gene vaccine for humans)include, but are not limited to, a bovine growth hormone polyadenylationsignal, a SV40 polyadenylation signal, and a LTR polyadenylation signal.Particularly, a SV40 polyadenylation signal present in pCEP4 plasmid(Invitrogen, San Diego, Calif.; called SV40 polyadenylation signal) isemployed.

In addition to control elements required for DNA expression, otherelements may be included in a DNA molecule. A HSV-derived promoter usedherein may further have an enhancer. The above-described additionalelements include an enhancer. An enhancer may be selected from the groupconsisting of human actin, human myosin, human hemoglobin, humanmuscular creatine and virus enhancers, such as enhancers from CMV, RSVand EBV, but is not limited to them.

A gene construct may be provided with a mammalian replication origin soas to maintain the construct outside a chromosome and produce a largenumber of copies within a cell. Plasmids pCEP4 and pREP4 (Invitrogen,San Diego, Calif.) include the replication origin of Epstein-Barr virusand a nuclear antigen EB NA-1 code region which causes replication oflarge numbers of copies of the episome. In some embodiments, cDNAencoding an immunomodulatory protein is inserted within pCDNA3.

In some preferred embodiments, a nucleic acid molecule including anucleotide sequence encoding the genes of a target protein, animmunomodulatory protein, and a protein which accelerates an immuneresponse to the target protein, is delivered. An example of such a geneis a gene encoding a cytokine or a lymphokine, such as α-interferon,gamma-interferon, platelet-derived growth factor (PDGF), TNF, epidermalgrowth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12. Insome embodiment, a gene construct used in a composition for immunizationpreferably includes the gene of GM-CSF.

To maximize protein production, a control sequence suitable for geneexpression in a cell to which a gene construct is inserted may beselected. Further, a codon which is most efficiently transcribed in acell may be selected. Those skilled in the art can easily produce a DNAconstruct which functions within a cell based on well-known techniques.

Such a nucleic acid can be obtained by a well-known PCR method, orchemically synthesized. These methods may be combined with a sitespecific mutagenesis method, a hybridization method, and the like.

The molecular biological methods, biochemical methods, andmicrobiological methods used herein are well known and commonly used inthe art, as disclosed in, for example, Ausubel F. A. et al Ed. (1988),“Current Protocols in Molecular Biology”, Wiley, New York, N.Y.;Sambrook J. et al., (1987) “Molecular Cloning: A Laboratory Manual”. 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Jikken Igaku [Experimental Medicine], “Experimental Methods for GeneIntroduction & Expression Analysis”, special issue, Yodo-sha, 1997; andthe like.

The term “homology” of a gene refers to the magnitude of identitybetween two or more gene sequences. Therefore, the greater the homologybetween two certain genes, the greater the identity or similaritybetween their sequences. Whether or not two genes have homology isdetermined by comparing their sequences directly or by a hybridizationmethod under stringent conditions. When two gene sequences are directlycompared with each other, the genes have homology if representatively atleast 50%, preferably at least 70%, more preferably at least 80%, 90%,95%, 96%, 97%, 98%, or 99% of the DNA sequence of the genes areidentical.

Comparison of identity between base sequences is herein calculated byBLAST which is a tool for analyzing a sequence using default parameters.

The term “polynucleotide hybridized under stringent conditions” as usedherein refers to a polynucleotide hybridized under well-known conditionscommonly used in the art. Such a polynucleotide can be obtained by acolony hybridization method, a plaque hybridization method, a Southernblotting hybridization method, or the like using a polynucleotideselected from the polynucleotides of the present invention as a probe.Specifically, such a polynucleotide can be identified by hybridizationusing a filter, on which DNA derived from a colony or a plaque isimmobilized, in the presence of 0.7 to 1.0 M NaCl at 65° C., followed bywashing the filter with SSC (saline-sodium citrate) solution having 0.1to 2-fold concentration (SSC solution with 1 fold concentration contains150 mM sodium chloride and 15 mM sodium citrate) at 65° C. Hybridizationcan be conducted in accordance with a method as described in anexperimental manual, such as Molecular Cloning 2nd ed., CurrentProtocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: CoreTechniques, A Practical Approach, Second Edition, Oxford UniversityPress (1995), and the like. Preferably, sequences hybridized understringent conditions herein exclude sequences consisting only of A andsequences consisting only of T.

The term “hybridizable polynucleotide” as used herein refers to apolynucleotide which can hybridize to another polynucleotide under theabove-described conditions for hybridization. Specific examples ofhybridizable polynucleotides include a polynucleotide having at least60% homology with the base sequence of DNA encoding a polypeptide havingan amino acid sequence indicated by SEQ ID NO: 2, 4 or 6, morepreferably a polynucleotide having at least 80% homology, and even morepreferably at least 95%. Homology described herein is represented bysimilarity indicated by a score using BLAST which is a search programemploying an algorithm developed by Altschul et al. (J. Mol. Biol. 215,403-410 (1990)).

The term “derived oligonucleotide” or “derived polynucleotide” refers toan oligonucleotide or polynucleotide including a derivative of anucleotide or having a linkage between nucleotides which is not normal.The above-described terms are interchangeably used. Specifically,examples of such an oligonucleotide include a derived oligonucleotide inwhich a phosphodiester bond is converted to a phosphothioate bond, aderived oligonucleotide in which phosphodiester bond is converted toN3′-P5′ phosphoramidate bond, a derived oligonucleotide in which riboseand phosphodiester bond are converted to peptide-nucleic acid bond, aderived oligonucleotide in which uracil is substituted with C-5 propynyluracil, a derived oligonucleotide in which uracil is substituted withC-5thiazole uracil, a derived oligonucleotide in which cytosine issubstituted with C-5 propynyl cytosine, a derived oligonucleotide inwhich cytosine is substituted with phenoxazine-modified cytosine, aderived oligonucleotide in which ribose is substituted with2′-O-propynyl ribose, a derived oligonucleotide in which ribose issubstituted with 2′-methoxyethoxy ribose, and the like.

The term “amino acid” as used herein refers to any naturally occurringamino acid and non-naturally occurring amino acid as described above.The term “derivated amino acid” as used herein refers to an amino acidwhich is different from naturally occurring amino acids but has afunction similar to that of its original naturally occurring amino acid.Such derivated amino acids are well known in the art.

The term “nucleotide” as used herein refers to any naturally occurringnucleotide and non-naturally occurring nucleotide. The term “derivednucleotide” as used herein refers to a nucleotide which is differentfrom naturally occurring nucleotides but has a function similar to thatof its original naturally occurring nucleotide. Such derived nucleotidesare well known in the art.

The term “biological activity” as used herein refers to the activitywhich a certain factor (e.g., polypeptide or protein) has within anorganism, including activity exhibiting various functions. For example,when the certain factor is an enzyme, its biological activity includesenzymatic activity. As another example, when the certain factor is aligand, its biological activity includes binding to a receptor to whichthe ligand corresponds.

The term “variant” as used herein refers to a substance, such aspolypeptide, polynucleotide, or the like, which differs partially fromthe original substance. Examples of such a variant include asubstitution variant, an addition variant, a deletion variant, atruncated variant, an allelic variant, and the like. The term “allele”as used herein refers to a genetic variant located at a locus identicalto a corresponding gene, where the two genes are distinguished from eachother. Therefore, the term “allelic variant” as used herein refers to avariant which has an allele relationship with a certain gene. The term“homolog” of a nucleic acid molecule as used herein refers to a nucleicacid molecule having a nucleotide sequence having homology with thenucleotide sequence of a reference nucleic acid molecule.Representatively, “homolog” refers to a polynucleotide which hybridizesto a reference nucleic acid molecule under stringent conditions. In thecase of the nucleic acid molecule of the present invention, a “homolog”is a nucleic acid molecule having a nucleic acid sequence havinghomology with a nucleic acid sequence encoding the amino acid sequenceof a protein, whose biological function is the same as or similar to thepromoter of the present invention. Therefore, the concepts of the terms“homolog” and “variant” overlap partially. Therefore, a homolog hasamino acid or nucleotide homology with a certain gene in a certainspecies (preferably at least 60% homology, more preferably at least 80%,at least 85%, at least 90%, and at least 95% homology). A method forobtaining such a homolog is clearly understood from the description ofthe present specification.

When introduction of a virus construct into a cell is herein required,the virus construct may be introduced into a cell by any method forintroducing DNA into a cell. Examples of such a method includetransfection, transduction, transformation, and the like (e.g.,electroporation, a method using a particle gun (gene gun), and thelike).

Pharmaceutical (medicinal) compositions herein preferably contain theherpes virus of the present invention and a pharmaceutically acceptablecarrier. Such pharmaceutical compositions are in any form as long as itis effective to a host. Examples of the form include, but are notlimited to, powders, granules, capsules, tablets, caplets, pills,solutions, troches, buccal tablets, sublingual tablets, vaginal tablets,elixirs, syrups, lemonades, suspensions, emulsions, aerosols,injections, infusions, nebulae, liniment, ointments, plasters, lotions,suppositories, and the like. Preferably, the present invention isprovided in the form of vaccine injections. The present invention may bein the form of sustained preparations. A DDS technique may be applied tothe present invention.

Preferably, the pharmaceutical composition of the present inventioncontains at least 10³ herpes viruses, and preferably at least 2×10³, atleast 5×10³, at least 10⁴, at least 2×10⁴, at least 5×10⁴, at least 10⁵,at least 2×10⁵, at least 5×10⁵, at least 10⁶, at least 2×10⁶, at least5×10⁶, at least 10⁷, at least 2×10⁷, at least 5×10⁷, at least 10⁸, atleast 2×10⁸, at least 5×10⁸, at least 10⁹, at least 2×10⁹, at least5×10⁹, at least 10¹⁰, or at least more than 10¹⁰ herpes viruses. Asuitable amount varies depending on circumstances. Those skilled in theart would determine the suitable amount, depending on the state of theactive ingredient in a composition, or in the case of a medicinalcomposition, on the conditions of a patient, the conditions of a diseaseof a patient, the administration route, dosage form, or the conditionsof a patient monitored during administration.

Examples of the pharmaceutically acceptable carrier include, but are notlimited to excipients, binders, disintegrants, lubricants, antioxidants,preservatives, colorants, flavoring agents, stabilizers, coating agents,diluents, emulsifiers, suspending agents, solvents, fillers, bulkyagents, buffers, delivery vehicles, and/or pharmaceutical adjuvants.

When the composition of the present invention is used as apharmaceutical composition, the composition further contains thefollowing medicinal ingredients:

central nerve system drugs (e.g., general anesthetics,sedative-hypnotics, anxiolytics, antiepileptics, anti-inflammatoryagents, stimulants, antihypnotics, antiperkinson agents, antipycohtics,combination cold remedies, and the like);

peripheral nerve agents (e.g., local anesthetics, skeletal musclerelaxants, autonomic nerve agents, antispasmodic agents, and the like);

sensory organ drugs (e.g., ophthalmological agents,otorhinolaryngological agents, antidinics, and the like):

circulatory organ drugs (e.g., cardiotonics, antiarrhythmics, diuretics,antihypertensive agents, vasoconstrictors, vasodilators,antihyperlipemia agents, and the like);

respiratory organ drugs (e.g., respiratory stimulants, antitussives,expectorants, antitussive extpectorants, bronchodilators, collutoriums,and the like);

digestive organ drugs (e.g., stegnotics, antiflatuents, peptic ulceragents, stomachics, antacids, cathartics, enemas, cholagogues, and thelike);

hormone agents (e.g. pituitary gland hormone agents, salivary glandhormone agents, thyroid gland hormone agents, accessory thyroid glandhormone agents, anabolic steroid agents, adrenal gland hormone agents,androgenic hormone agents, estrogen agents, progesterone agents, mixedhormone agents, and the like);

urogenital organ and anus drugs (e.g., urinary organ agents, genitalorgans agents, uterotonics, hemorrhoids agents, and the like);

dermatologic drugs (e.g., dermatologic disinfectants, wound protectingagents, pyogenic diseases agents, analgesics, antipruritics,astringents, antiphlogistics, parasitic skin diseases agents,emollients, hair agents, and the like);

dental and oral agents;

drugs for other organs;

vitamin agents (e.g., vitamin A agents, vitamin D agents, vitamin Bagents, vitamin C agents, vitamin E agents, vitamin K agents, mixedvitamin agents, and the like):

nutritive agents (e.g., calcium agents, inorganic preparations,saccharides agents, protein amino acid preparations, organ preparations,infant preparations, and the like);

blood and body fluid drugs (e.g., blood substitute agents, styptics,anticoagulants, and the like):

dialysis drugs (e.g., kidney dialysis agents, peritonea dialysis agents,and the like);

other metabolic drugs (e.g., organ disease agents, antidotes, antabuses,arthrifuges, enzyme preparation, diabetic agents, and others);

cell activating agents (e.g., chlorophyll preparations, pigment agents,and the like);

tumor agents (e.g., alkylation agents, antimetabolites, antineoplasticantibiotic preparations, antineoplastic plant extract preparations, andthe like);

radiopharmaceuticals;

allergy drugs (e.g., antihistamic agents, irritation therapy agents,non-specific immunogen preparations, and other allergy drugs, crudedrugs and drugs based on Chinese medicine, crude drugs, Chinese medicinepreparation, and other preparations based on crude drug and Chinesemedicine formulation);

antibiotic preparations (e.g., acting for gram-positive bacteria,gram-negative bacteria, gram-positive mycoplasmas, gram-negativemycoplasmas, gram-positive rickettsia, gram-negative rickettsia,acid-fast bacteria, molds, and the like);

chemotherapeutic agents (e.g., sulfa drugs, antitubercular agents,synthetic antimicrobial agents, antiviral agents, and the like);

biological preparations (e.g., vaccines, toxoids, antitoxins, leptospireantisera, blood preparations, biological test preparations, and otherbiological preparations, and antiprotozoal drugs, anthelmintics, and thelike);

dispensing agents (e.g., excipients, ointment bases, solvents, flavors,colorants, and the like);

diagnostic drugs (e.g., contrast media, function testing reagents, andthe like);

sanitation drugs (e.g., preservative):

xenodiagnostic drugs (e.g., cytologic examination drugs, and the like);

non-categorized drugs which do not aim mainly for therapy; and

narcotics (e.g., opium alkaloid drugs, coca alkaloid preparations,synthetic narcotics, and the like).

The pharmaceutical composition of the present invention can be easilyproduced by those skilled in the art with reference to JapanesePharmacopeia, United States Pharmacopoeia, Pharmacopoeias of othercountries, Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, ed., Mack Publishing Company, 1990, or the like.

When the present invention is prescribed as a pharmaceuticalcomposition, it may be parenterally administered. Alternatively, such acomposition may be intravenously or subcutaneously administered. Whenadministered systemically, therapeutic compositions for use in thepresent invention maybe in the form of orally acceptable aqueoussolution which does not contain a pyrogen. Such a pharmaceuticallyacceptable aqueous solution maybe prepared by techniques in the art ifpH, isotonicity, stability, and the like are carefully controlled.

In a preferred embodiment, the HSV of the present invention or apharmaceutical composition containing the same may be administered inthe form of inoculation. The pharmaceutical composition containing theattenuated HSV of the present invention is not limited to a particularinoculation form and can be used in any known form. Preferably, such acomposition is inoculated as an injection of aqueous solution orsuspension to a patient. Such an injection includes infusion solution,supplements, and the like.

The above-described injection may contain a commonly used additive inaddition to the attenuated HSV of the present invention. The additive isnot particularly limited. For example, the injection may optionallycontain a surfactant, an emulsifier, suspending agents, a preservative,a soothing agent, a stabilizer, or the like as an ingredient. Examplesof the surfactant include Tween80, polyoxyl 40 stearate, sorbitansesquioleate, glyceryl monostearate, lauromacrogol, and the like.Examples of the emulsifier include gum arabic, traganth, sodiumalginate, and the like. Examples of the suspending agents includealuminum monostearate, carboxymethyl cellulose, methyl cellulose, andthe like. Examples of the preservative include phenol, phenylmercuricnitrate, benzalkonium chloride, benzethonium chloride, benzyl alcohol,chlorobutanol, and the like. Examples of the soothing agent includebenzyl alcohol, chlorobutanol, sorbitol, and the like. Examples of thestabilizer include buffering agents, such as citric acid, acetic acid,tartaric acid, succinic acid, and the like, propylene glycol, diethylin,sulfite, ascorbic acid, Rongalite, and the like.

Further, water, physiological saline, dextrose, glycerol, ethanol,propylene glycol, polyethylene glycol, vegetable oil, organic ester suchas ethyl oleate and the like, pH buffering agents, adjuvant, immuneactivators for enhancing the effect of attenuated HSV, and the like maybe added.

In the present invention, a method for producing the above-describedinjections are not particularly limited. The injections may be producedby a commonly used device and method. The prepared injections may besubjected to bacteria elimination by filtration, and optionally tolyophilization and the like. The injections are administeredsubcutaneously, intramuscularly, intravenously, intratumorally,intraperitoneally, intratracheally, intravesically, intraintestinally,intrarectally, intrabuccally, intraocularly, intraarterially, or thelike by a commonly used method or by infusion.

The above-described injections may be dissolved into solutionimmediately before administration. The injections may be prepared in theform of a solid.

The accurate doses of individual pharmaceutical compositions containingthe attenuated HSV of the present invention vary depending on anadministration method, differences between individual subject patients,types of diseases, the conditions of subject patients, and are notnecessarily determined in the same manner. However, for example, whenadministered to a human as an injection, the dose of attenuated HSV isabout 0.01 ng to 10 mg/kg a day at one or several dosage times for anadult. Preferably, administration may be conducted while monitoring theconditions of a patient if necessary.

The attenuated HSV of the present invention can be used in conjunctionwith a prodrug in accordance with a known method. The administrationroute and dose of the prodrug are determined in accordance with theknown method. The prodrug can be used simultaneously or alternately withthe attenuated HSV of the present invention.

The present invention may also be administered preferably in the form ofa vaccine. Vaccine means an antigen in any of various-forms (e.g.,protein, DNA, and the like) which is used to prevent (or treat) acertain type of disease (e.g., contagious diseases, infectious diseases,and the like). Attenuated live pathogens (live vaccine), inactivepathogens (or a part thereof), metabolites of a pathogen (toxin,inactivated toxin (i.e., toxoid), or the like), DNA vaccines, or thelike are used depending on the type of infection, transmission,epidemic, or the like. Vaccination actively develops immunity (humoralimmunity, cell-mediated immunity, or both) within the body of organisms(humans, livestock, and vectors) and prevent the infection,transmission, epidemic, or the like of pathogens.

The vaccines of the present invention are not particularly limited toany dosage form, and are prepared in accordance with methods per seknown in the art. The vaccines of the present invention are preferablylive vaccines containing the above-described HSV gene recombinant.Further, the vaccines of the present invention may be in the form of anemulsion containing various adjuvants. The adjuvants aid sustenance of ahigh level of immunity when the above-described HSV gene recombinant isused in a smaller dose than when it is used alone. Examples of theadjuvants include Freund's adjuvant (complete or incomplete), adjuvant65 (including peanut oil, mannide monooleate and aluminum monostearate),and aluminum hydrate, aluminum phosphate or mineral gel such as alum.For vaccines for humans-or edible animals, adjuvant 65 is preferable.For vaccines for commercial animals, mineral gel is preferable.

In addition to the above-described adjuvants, the vaccines of thepresent invention may contain at least one additive for preparationsselected from diluents, aroma chemicals, preservatives, excipients,disintegrants, lubricants, binders, surfactants, plasticizers, and thelike.

The administration routes of the vaccines of the present invention arenot particularly limited. However, the vaccines are preferablyadministered parenterally (e.g., intravenously, intraarterially,subcutaneously, intradermal, intramuscularly or intraperitoneally).

The dose of the vaccines of the present invention can be selecteddepending on various conditions: whether administration is intended toprevent or treat diseases caused by HSV infection: whether infection isprimary or recurrent: the age and weight, conditions of patients; theseverity of disease; and the like. When intended to treat diseasescaused by recurrent infection, the dose of the vaccines of the presentinvention can be preferably about 0.01 ng to 10 mg per kg weight, morepreferably about 0.1 ng to 1 mg/kg.

The number of administrations of the vaccines of the present inventionvaries depending on the above-described various conditions, and is notnecessarily determined in the same manner. However, preferably, thevaccines are repeatedly administered at the intervals of days or weeks.Particularly, administration is conducted at a total of several times,or preferably about one to two times, at the intervals of about 2 to 4weeks. The number of administrations (administration time) is preferablydetermined by symptomatology or a fundamental test using antibody titerwhile monitoring the conditions of diseases.

The term “diseases or disorders” as used herein refers to diseases ordisorders to which the virus constructs of the present invention areeffective. Examples of the diseases or disorders include herpesvirus-related diseases or disorders (e.g., gingivostomatitis, herpesfacialis, herpes labialis, herpes keratoconjunctivitis, herpesencephalitis, genital herpes, neonatal herpes, chickenpox, herpeszoster, infectious mononucleosis, Burkitt's lymphoma, rhinopharyngealcancer, pneumonia, cytomegalic inclusion disease, exanthema subitum,pneumonia, Kaposi's sarcoma, PEL, Castleman's disease), cancer (e.g.,ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, urethracancer, colon cancer, skin cancer, malignant melanoma, osteosarcoma,head and neck squamous cell carcinoma, stomach cancer, prostate cancer,breast cancer, lung cancer, colon cancer, lymphoma, hepatoma,mesothelioma, melanoma, astrocytoma, oligodendroglioma, meningioma,neurofibroma, glioblastoma, ependymoma, schwannoma, neurofibrosarcoma,medulloblastoma, fibrosarcoma, squamous cell carcinoma, neuroectodermcancer, thyrocele, pituitary gland tumor, epidermoid carcinoma, and thelike), other diseases or disorders (e.g., diseases or disorders causedby other viruses (e.g., HIV, influenza virus, rotavirus, and the like)or bacteria (e.g., Bordetella pertussis, Corynebacterium diphtheriae,Clostridium tetani, and the like)), prokaryotes (e.g., gonococcus,Listeria monocytogenes, dysentery bacillus, and the like) and eukaryotes(e.g., unicellular pathogens, multicellular parasites, and the like).

Therefore, the herpes simplex virus of the present invention and acomposition containing the same can be used to prevent or treat canceras described above. Preferably, the herpes simplex virus of the presentinvention and a composition containing the same may be applied toprogressive pancreatic cancer, ovarian cancer, and the like whichusually occur with peritoneal dissemination and are difficult to treatby surgical excision or chemotherapy, and to solid tumor. The presentinvention may be used to immunize an individual against at least oneform of cancer. Particularly, the present invention may be used toprecautiously immunize a human individual having a disposition to sufferfrom a particular cancer or a human individual who has previously hadcancer and therefore has the risk of recurrence. The development ofgenetics, technology and epidemiology makes it possible to determine thepossibility and risk assessment of the occurrence of cancer forindividuals. With gene screening and/or a family health history, it ispossible to predict the possibility of the occurrence of any of severaltypes of cancers for individuals.

Pharmaceutical compositions containing the attenuated HSV of the presentinvention may be prepared by combining an effective amount of theattenuated HSV with an appropriate medicinal carrier and other additivesby a commonly used method. “Effective amount” refers to the amountnecessary or sufficient to exhibit an intended effect with little sideeffect, although it varies depending on the type or usage form thereofand is not necessarily determined in the same manner.

The pharmaceutical compositions containing the attenuated HSV of thepresent invention can be inoculated to patients in known forms,preferably injections of aqueous solution or suspension. The inoculationforms are not particularly limited. The injections include infusionsolution, nutritive replenishing solution, and the like.

The injections may contain a commonly used additive in addition to theattenuated HSV of the present invention. Examples of the additiveinclude, but are not particularly limited to, surfactants, emulsifiers,suspending agents, preservatives, soothing agents, stabilizers, and thelike, which are optionally used. Examples of the surfactants includeTween80, polyoxyl 40 stearate, sorbitan sesquioleate, glycerylmonostearate, lauromacrogol, and the like. Examples of the emulsifiersinclude gum arabic, traganth, sodium alginate, and the like. Examples ofthe suspending agents include aluminum monostearate, carboxymethylcellulose, methyl cellulose, and the like. Examples of the preservativesinclude phenol, phenylmercuric nitrate, benzalkonium chloride,benzethonium chloride, benzyl alcohol, chlorobutanol, and the like.Examples of the soothing agents include benzyl alcohol, chlorobutanol,sorbitol, and the like. Examples of the stabilizers include bufferingagents (e.g., citric acid, acetic acid, tartaric acid, succinic acid,and the like), propylene glycol, diethylin, sulfite, ascorbic acid,Rongalite, and the like.

Further, water, physiological saline, dextrose, glycerol, ethanol,propylene glycol, polyethylene glycol, vegetable oil, organic ester suchas ethyl oleate and the like, pH buffering agents, adjuvants, immuneactivators for enhancing the effect of attenuated HSV, and the like maybe added.

In the present invention, a method for producing the above-describedinjections are not particularly limited. The injections may be producedby a commonly used device and method. The prepared injections may besubjected to bacteria elimination by filtration, and optionally tolyophilization and the like. The injections are administeredsubcutaneously, intramuscularly, intravenously, intratumorally,intraperitoneally, intratracheally, intravesically, intraintestinally,intrarectally, intrabuccally, intraocularly, intraarterially, or thelike by a commonly used method or by infusion.

The above-described injections maybe dissolved into solution immediatelybefore administration. The injections may be prepared in the form of asolid.

The accurate doses of individual pharmaceutical compositions containingthe attenuated HSV of the present invention vary depending on anadministration method, differences between individual subject patients,types of diseases, the conditions of subject patients, and are notnecessarily determined in the same manner. However, for example, whenadministered to a human as an injection, the dose of attenuated HSV isabout 0.01 ng to 10 mg/kg a day at one or several dosage times for anadult. Preferably, administration may be conducted while monitoring theconditions of a patient if necessary.

The attenuated HSV of the present invention can be used in conjunctionwith a prodrug in accordance with a known method. The administrationroute and dose of the prodrug are determined in accordance with theknown method. The prodrug can be used simultaneously or alternately withthe attenuated HSV of the present invention.

The term “prodrug” as used herein refers to a drug which is inactive asit is and becomes active when it is chemically changed in the body by adrug-metabolizing enzyme (e.g., purine and pyrimidine derivatives usedas chemotherapeutic agents for cancer). Examples of the prodrugs hereinpreferably include ganciclovir, acyclovir, taxol, camptothecin, guaninenucleoside derivatives (e.g., A-5021), and the like. A prodrug hereinpreferable for the present invention is a prodrug which is converted toan active form by a suicide gene contained in the attenuated HSV of thepresent invention.

The term “suicide gene” as used herein refers to a gene which can killthe cell in which it is expressed. Representatively, such a gene is ametabolically toxic gene. For example, a method for introducing asuicide gene incorporated into a virus construct into tumor cells todrive them to suicide is herein exemplified. Specifically, thymidinekinase may be transduced into herpes simplex virus.

The term “ability to select cancer cells” as used herein indicates thatthe growth rate in cancer cells is higher than in normal cells.

The present invention may be provided in conjunction with animmunomodulatory protein. The term “immunomodulatory protein” as usedherein refers to proteins and nucleic acid molecule expression productswhich accelerate and/or regulate immune responses. Therefore, in apreferred embodiment, the immunomodulatory proteins may be delivered asan immunotherapeutic agent, or an ingredient of a vaccine.

Examples of the immunomodulatory proteins include chemokines, adhesivemolecules, cytokines, co-stimulatory molecule, growth factors, andreceptor molecules. The chemokines include MIP-1α, MIP-1β, RANTEs, IL-8and MCP-1. Examples of the adhesive molecules include selectin familyconstructs, mucin-like molecules, integrin family constructs, andimmunoglobulin superfamily constructs. Examples of the select in familyconstructs include L-selectin, P-selectin, and E-selectin. Themucin-like molecules are ligands for the selectin family constructs.Examples of the mucin-like molecule include CD34, GlyCAM-1, andMadCAM-1. Examples of the integrin family constructs include LFA-1,VLA-1, Mac-1, and p150.95. Examples pf the immunoglobulin superfamilyconstructs include PECAM-1, ICAMs (ICAM-1, ICAM-2, and ICAM-3), CD2, andLFA-3. Examples of the cytokine include mutants of M-CSF, GM-CSF, G-CSF,CSF, IL-4, and IL-18 (including deletion of the first about 35 aminoacid residues residues which are present in the precursor of a proteinbut are not present in the protein in the mature form). Examples ofco-stimulatory molecules include CD40 and CD40 ligands (CD40L). Examplesof growth factors include IL-7, nerve growth factors, and a vascularendothelial growth factor. Examples of the receptor molecules include aFas lethal gene expression product, a tumor necrosis factor TNFreceptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. The compositions of thepresent invention may contain caspase (ICE).

In one embodiment of the present invention, the immunomodulatoryproteins are delivered by administrating the nucleic acid moleculeswhich are expressed into the immunomodulatory proteins when taken intocells. In some embodiments of the present invention, theimmunomodulatory proteins are delivered by administrating the proteinsper se. In some embodiments of the present invention, theimmunomodulatory proteins are delivered by administrating the nucleicacids or the proteins per se. In one embodiment of the presentinvention, the immunomodulatory proteins are delivered by administratingthe nucleic acids and the proteins per se simultaneously.

In some embodiments of the present invention, the above-describedimmunomodulatory proteins (as proteins or nucleic acid moleculesencoding the proteins) are administered as a supplement along with acomposition or otherwise a vaccine composition. In this case, thevaccine is any of a subunit, an inactivated vaccine, an attenuated livevaccine, a cellular vaccine, a recombinant vaccine, and a nucleic acidor DNA vaccine. In the case of the attenuated live vaccine, the cellularvaccine, the recombinant vaccine, or the nucleic acid or DNA vaccine,the immunomodulatory proteins are encoded by the nucleic acid moleculesof the vaccines.

Compositions (e.g., vaccine) are herein provided for treating orpreventing pathogens other than herpes virus (e.g., viruses (e.g., HIV,influenza virus, rotavirus, and the like), or bacteria). Suchcompositions comprise at least one gene of the pathogen which isincluded in an attenuated herpes virus as an exogenous gene. Theexogenous gene preferably is full length but may be a partial sequenceas long as it contains at least an epitope capable of triggeringimmunity. The term “epitope” as used herein refers to an antigenicdeterminant whose structure has been revealed. A method for determiningan epitope is known in the art. Once the primary nucleic acid or aminoacid sequence of a protein is provided, such epitopes can be determinedby such a known routine technique. A useful epitope may have at least alength of three amino acids, preferably, at least 4 amino acids, 5 aminoacids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10amino acids, 15 amino acids, 20 amino acids, or 25 amino acids.

Examples of such an exogenous gene include, but are not limited to,gp120, gp41 and gp17 matrix proteins, p24 capsid protein, reversetranscriptase (HIV pol), tat rev, and the like in the case of HIV; VP4,VP6 and VP7 and the like in the case of rotavirus, and HA, NA and NP andthe like in the case of influenza virus.

The immunomodulatory proteins are useful in inducing and acceleratingcytotoxic T lymphocyte (CTL) responses and/or antibody responses, and/orT lymphocyte growth responses.

The immunomodulatory proteins which induce and accelerate CTL responsesare particularly useful when administered in conjunction with or as apart of vaccines against pathogens inside cells, autoimmune diseases, orcancers. The immunomodulatory protein which induce and accelerate CTLresponses are particularly useful when administered along withattenuated live vaccines, cellular vaccines, recombinant vaccines, andnucleic acid or DNA vaccines. Alternatively, the immunomodulatoryprotein which induce and accelerate CTL responses are useful as animmunotherapeutic agent which is administered for patients with canceror intracellular infection. The immunomodulatory protein which induceand accelerate CTL responses are useful when administered toimmunocompromised patients.

Subject hosts of diseases may be herein any animal which can be injectedwith herpes virus. Examples of such animals include primates (e.g.,monkeys and humans), cattles (e.g., beef cattle and dairy cattle),horses, pigs, cats, dogs, chickens, and the like. The hosts arepreferably primates, and more preferably humans.

The age of subject hosts may be herein any age at which the subjecthosts can be infected with herpes virus. For example, the subject hostsinclude the elderly, adults, children, babies, fetuses, and the like.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides herpes virus in which a non-essentialgene for replication is inactivated. More specifically, the presentinvention provides herpes virus in which a non-essential gene forreplication present in the UL or US region is inactivated.Conventionally, herpes virus in which a gene in an inverted repeatregion is modified to transform its characteristic is used. However, itcannot be said that there is a herpes virus which is clinicallyeffective to diseases caused by herpes virus or cancer. Therefore, byinactivating a non-essential gene for replication in the UL or USregion, desired characteristics (e.g., attenuation, selectivity tocancer cells, an increase in safety for hosts (e.g., humans), and thelike) can be obtained, resulting in utility advantageous overconventional techniques.

In one embodiment, the vaccines of the present invention arecharacterized by containing a HSV gene recombinant which does notexpress US3. Examples of such a recombinant include a gene recombinantin which among the HSV genes, the US3 gene is treated so as not to betranslated; a gene recombinant in which among the HSV genes, the US3gene is subjected to recombination, and when the recombined gene istranslated, a protein different from US3 is expressed; and the like.

A gene recombination process for preparing the above-described generecombinant is not particularly limited. For example, a part or entiretyof the US3 gene is deleted; a part or entirety of the US3 gene issubstituted; a part of the US3 gene is inverted, a part of the US3 geneis repeated; a part of the US3 gene is translocated; a gene fragment isinserted in the US3 gene so as to interrupt the US3 gene; and the like.Among them, deleting a part or entirety of the US3 gene, substituting apart or entirety of the US3 gene, or inserting a gene fragment in theUS3 gene so as to interrupt the US3 gene is preferable. Particularly,inserting a gene fragment in the US3 gene so as to interrupt the US3gene is more preferable. Even more preferably, the gene fragment to beinserted contains a signal which stops translation. As a signal whichstops translation, a polyadenylation signal derived from SV40 ispreferable.

The above-described gene recombination process is well established inthe art and therefore can be easily conducted in accordance with, forexample, a commercially available experimental manual, e.g., MolecularCloning (Cold Spring Harbor Laboratory), issued in 1982; MolecularCloning, 2nd ed. (Cold Spring Harbor Laboratory), issued in 1989; or thelike.

The “HSV gene recombinant which does not express US3” used in thepresent invention includes L1BR1. L1BR1 is prepared using HSV-2 strain186. This preparation method will be described below with reference toFIG. 10.

FIG. 10A shows the gene of the HSV-2 strain 186, indicating the mapunits. FIG. 10B shows a Hind III cleavage map of the strains. As can beseen from FIGS. 10C and 10D, the US3 gene is present in a Hind III Lfragment of 9.6 kb which is the US region (FIG. 10B). According to thebase information of the US region, the Bgl II cleavage site in the9.6-kb Hind III L fragment (G in FIG. 10C) is contained in the US3 gene(FIGS. 10C and 10D). Therefore, with the Bgl II cleavage site, the US3gene is inactivated, thereby preparing a US3-deficient HSV.

It should be noted that it is known that the restriction endonucleasecleavage pattern (FIG. 10C) of the HSV-2 strain 186 by Bam HI, Xba I,Bal II matches that. of a HSV-2 strain HG52 (McGeoch et al., 1987).

More specifically, a 4.8-kb Hind III-Xba I fragment is preliminarilycloned from the gene of the HSV-2 strain 186. A method for preliminarycloning maybe conducted using a known cloning vector like pBluescriptsuch as pLHX and the like in accordance with a method per se known.

Thereafter, the above-described preliminarily cloned Hind III-Xba Ifragment is digested with Bgl II, so that a reading frame of 1443 bp iscleaved at a 215th residue from the 5′ end. A 4.3-kb gene fragment shownin FIG. 10E is inserted into the cleavage site. Specifically, the genefragment shown in FIG. 10E is a 4.3-kb Bam HI fragment containing a geneencoding lacZ fused with a HSV-1 β8 promoter at the 5′ end and apolyadenylation signal derived from SV40 at the 3′ end. Thepolyadenylation signal derived from SV40 stops the translation of thelacZ gene (Spaete and Mocarski, 1985; and Ho and Mocarski, 1988).

The resultant gene having the US3 gene interrupted by the lacZ gene,i.e., a gene in which a gene fragment indicated by E at position “G” inthe gene fragment shown in FIG. 10C, is cloned by a known method. Itshould be noted that a gene recombinant obtained by inserting theabove-described gene into a known cloning vector pLHX is referred to aspHZL1.

Thereafter, Vero cells are cotransfected with the complete DNA of theinfectious HSV-2 strain 186 and the DNA of the linear pHZL1 containingthe US3 gene interrupted by the lacZ gene (simultaneous introductioninto cells). The two simultaneously introduced genes are recombined, theHSV gene recombinant is multiplied in the Vero cells. The HSV generecombinant containing the US3 gene interrupted by the lacZ gene can beselected by the colors of plaques stained by Xgal staining.Specifically, since the lacZ gene encodes β-galactosidase, if the HSVgene recombinant contains the lacZ gene, β-galactosidase is expressed.This enzyme reacts with its substrate Xgal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) to produce a darkblue degradation products. Therefore, by selecting blue plaques, the HSVgene recombinant containing the lacZ gene can be selected. One of theselected gene recombinants is purified by the selection of a blueplaque, followed by further five rounds of plaque cloning. The purifiedgene recombinant is grown in Vero cells to prepare L1BR1.

Whether or not the above-described gene recombinant L1BR1 contains thelacZ gene at a site which interrupts the US3 gene can be determined bycomparing L1BR1 with the gene of a wild-type HSV-2 strain 186.Specifically, the L1BR1 gene is treated with a restriction enzyme,followed by Southern blotting hybridization analysis using a 1.6-kb BamHI fragment in the Hind III L fragment as a probe. The gene of thewild-type HSV-2 strain 186 is treated in the same manner. As a result,if the 1.6-kb fragment which is observed in the wild-type gene is lostin the L1BR1 gene, it can be confirmed that the lacZ gene interrupts theUS3 gene.

Preferably, the attenuated HSV of the present invention has ability toselect cancer cells, i.e., can be multiplied favorably in cancer cells.

In another embodiment, as an attenuated HSV in which at least oneaccessory gene is inactivated, UL39 or UL40 maybe in activated.Particularly, an attenuated HSV in which UL39 or UL40, and at least oneother accessory gene are inactivated is preferable.

The attenuated HSV in which UL39 or UL40 is inactivated must rely on ahost for ribonucleotide reductase which is encoded by UL39, and UL40 andis necessary for DNA and deoxyribonucleotide metabolism. Therefore, whenthe attenuated HSV in which UL39 or UL40 is inactivated is inoculatedinto cancer patients, the attenuated HSV can be multiplied favorably inactively dividing cancer cells. The cancer cells infected with theattenuated HSV are preventing from growing due to the cytopathic effectof the virus, potentially leading to death.

When at least one other accessory gene is inactivated in addition toUL39 or UL40, the pathogenicity of the attenuated HSV can be furtherreduced while maintaining the favored growth in cancer cells.

Examples of the attenuated HSV in which UL39 or UL40 and at least oneother accessory gene are inactivated include an attenuated HSV in whichUL39 (or UL40), UL55 and UL56 are inactivated, an attenuated HSV inwhich UL39 (or UL40) and UL2 are inactivated, an attenuated HSV in whichUL39 (or UL40) and US3 are inactivated, an attenuated HSV in which UL39(or UL40), UL2 and US3 are inactivated, and the like.

The attenuated HSV in which UL39 (or UL40), UL55 and UL56 areinactivated and the attenuated HSV in which UL39 (or UL40) and UL2 areinactivated have a higher level of safety than that of the attenuatedHSV in which only UL39 (or UL40) is inactivated. The attenuated HSV inwhich UL39 (or UL40) and US3 are inactivated and the attenuated HSV inwhich UL39 (or UL40), UL2 and US3 are inactivated can cause apoptosis ina cancer cell as well as having a high level of ability to select cancercells.

The vaccines of the present invention are useful as an agent forpreventing and treating the above-described diseases caused by HSV.Particularly, the vaccines of the present invention are useful for anagent for preventing and treating sexually transmitted diseases (e.g.,genital herpes) caused by HSV.

By administration of the vaccines of the present invention, the severityand/or infection rate of HSV infection can be reduced.

As can be seen from the Examples described below, a US3-deficient HSVgene recombinant contained in the vaccines of the present inventionincreases the number of Fas⁺ mononuclear cells, and quickly activatesdendritic cells, macrophages, and T cells. Further, the recombinantincreases IL-12 which is an antiviral cytokine, and IFN-γ (interferonγ). IL-12 and IFN-γ stimulate and activate TH1 cells which in turnactivate cell-mediated immunity. Thus, by administration of the vaccinesof the present invention containing the US3-deficient HSV generecombinant, an immune function against HSV infection is activated, sothat the severity and/or infection rate of HSV infection can be reducedas described above.

Recently, it is indicated that the US3 gene of herpes simplex virus type1 (hereinafter referred to as HSV-1) and HSV-2 is necessary forinhibition of apoptosis in infected cells (Leopardi, R., Van Sant, andRoizman B. (1997), Proc. Natl. Acad. Sci. USA. 94, 7891-7896; Asano, S.,Honda, T., Goshima, F., Watanabe, D., Miyake, Y., Sugiura, Y., andNishiyama, Y. (1999), J. Gen. Virol. 80, 51-56). Therefore, theUS3-deficient HSV gene recombinant has an advantage of lackinginhibition of apoptosis in infected cells. Specifically, when theUS3-deficient HSV gene recombinant is administered, infected cells areimmediately driven to apoptosis, so that the US3-deficient HSV generecombinant is suppressed from being excessively multiplied. Moreover,the infected cells which has caused apoptosis play a role in antigenpresentation, thereby further enhancing activation of immunity againstHSV infection.

In another embodiment, in the attenuated herpes simplex virus of thepresent invention (hereinafter also referred to as attenuated HSV), outof non-essential genes for replication, the US3 gene, and/or the UL56gene, which is a pathogenicity-related gene, are inactivated by deletinga part or entirety of the DNA base sequences thereof, or by basesequence substitutions, modifications, insertions, or the like.Alternatively, inactivation can be carried out by controllingtranscription of the US3 gene and/or the UL56 gene.

One attenuated HSV used in the present invention is clone 10 (MNO10)which is one of clones collected from strain HF. This clone has thegenome of herpes virus in which 3832 bps are deleted at positions 116515to 120346, and 136 amino acids at the N terminus side out of a total of197 amino acids of UL56, and its upstream region are deleted. MNO10 ispreserved and distributed by the Laboratory of Virology, ResearchInstitute for Disease Mechanism and Control, Nagoya University School ofMedicine in accordance with the requirements under the Japanese PatentLaw and is easily available.

The attenuated HSV of the present invention may be prepared using HSV-1or alternatively HSV-2.

Examples of a method for inactivating the US3 and/or UL56 genes of theattenuated HSV include, but are not limited to, insertion of anothersequence, removal of a part of a sequence, modification of a base,substitution of a base, and the like, which are known methods.

By inactivating the US3 gene and/or the UL56 gene, the attenuated HSV ofthe present invention can be multiplied favorably in cancer cells.Further, a suicide gene can be effectively expressed in targeted cancercells, potentially leading to an effective anticancer function.

The attenuated herpes simplex virus of the present invention in whichthe US3 gene and/or the UL56 gene are inactivated is combined with aprodrug which is converted to an active form by the herpes virus, or isused alone without such a combination, to treat cancer.

In one preferred embodiment, the attenuated HSV of the present inventioncontains the thymidine kinase gene. In this case, if ganciclovir whichis a prodrug is administered, the ganciclovir is converted to an activeform by phosphorylation by thymidine kinase, thereby inhibiting DNApolymerase in cancer cells. Further, the attenuated HSV is taken intoDNA, thereby inhibiting DNA and deoxyribonucleotide metabolism andDNA-independent RNA synthesis. A gene having such a function is calledsuicide gene. In the present invention, an exogenous suicide gene may beexpressibly incorporated in addition to the above-described thymidinekinase gene.

The above-described exogenous suicide gene is, but not particularlylimited to, any gene that can convert a prodrug to an active form.Examples of the exogenous suicide gene include a cytosine deaminasegene, the gpt gene of E. coli, the deoD gene of E. coli, acarboxyesterase gene, and the like.

In another preferred embodiment, the present invention provides aUS3-deficient HSV-2 virus.

The present invention provides an improved method for immunizing anindividual comprising the step of delivering a gene construct to cellsin the individual as a part of a vaccine composition (including DNAvaccines, attenuated live vaccines, and recombinant vaccines). The geneconstruct comprises a nucleotide sequence which encodes animmunomodulatory protein and is operatively linked to a control sequencewhich can function in the vaccine so as to achieve expression of thegene construct. The improved vaccine causes an enhanced cell-mediatedimmune response.

The attenuated herpes simplex virus of the present invention in whichthe US3 gene and/or the UL56 gene are inactivated is combined with aprodrug which is converted to an active form by the herpes virus, or isused alone without such a combination, to treat cancer.

Examples of the prodrugs herein preferably include ganciclovir,acyclovir, taxol, camptothecin, and the like. A prodrug hereinpreferable for the present invention is a prodrug which is converted toan active form by a suicide gene contained in the attenuated HSV of thepresent invention.

The attenuated HSV of the present invention contains the thymidinekinase gene. In this case, if ganciclovir which is a prodrug isadministered, the ganciclovir is converted to an active form byphosphorylation by thymidine kinase, thereby inhibiting DNA polymerasein cancer cells. Further, the attenuated HSV is taken into DNA, therebyinhibiting DNA and deoxyribonucleotide metabolism and DNA-independentRNA synthesis. A gene having such a function is called suicide gene. Inthe present invention, an exogenous suicide gene may be expressiblyincorporated in addition to the above-described thymidine kinase gene.

The above-described exogenous suicide gene is, but not particularlylimited to, any gene that can convert a prodrug to an active form.Examples of the exogenous suicide gene include a cytosine deaminasegene, the gpt gene of E. coli, the deoD gene of E. coli, acarboxyesterase gene, and the like.

EXAMPLE

Hereinafter, the present invention will be described by way of examples.The following examples are provided only for illustration purposes.Therefore, the present invention is not limited to the examples. Thescope of the present invention is limited only by the appended claims.It should be noted that % indicates % by weight unless otherwisementioned.

Example 1

Preparation of Attenuated HSV (UL39-inactivated HSV)

A DNA fragment of HSV-1 containing the UL39 gene was incorporated intoplasmid pBluescript (manufactured by Stratagene) for cloning, so as toprepare DNA having a lacZ cassette interrupting the open reading frameof the UL39 gene. In this case, the lacZ cassette is a DNA fragmentcontaining a promoter for the HSV UL39 at the 5′ end thereof and thelacZ gene of E. coli and the polyadenylation signal of SV40 downstreamof the promoter. Infectious HSV DNA was prepared from cells infectedwith HSV. Cells were transfected with this DNA. After three days, theproduced viruses were collected. A plaque which was stained blue in thepresence of X-gal was collected. Further plaque cloning was conductedthree times. The resultant viruses were multiplied in Vero cells andthen stocked. The thus-obtained mutant was subjected to Southern blot,PCR, Western blot, and the like, so that it was confirmed that UL39 wasinactivated.

Example 2

Preparation of Attenuated HSV (Hh) in which UL39 and UL56 Genes notInvolved in DNA and Deoxyribonucleotide Metabolism are Inactivated

There is provided a strain HF derived from HSV-1 in which a regioncontaining the UL56 gene is deficient (kindly provided by Dr. ShinIsomura, Department of Pediatrics, Nagoya University School ofMedicine). The pathogenicity of this strain is significantly low wheninoculated intraperitoneally in a mouse as compared to a wild type ofthe strain. Further, the strain causes cell fusion with infected cells.Vero cells were infected with the above-described UL39-deficient virusand the strain HF. Among the progeny viruses, viruses which cause cellfusion and produce blue plaque in the presence of X-gal were collected.After cloning the viruses, it was confirmed by PCR and Western blot thatUL39 and UL56 are deficient in the viruses.

Example 3

Preparation of Strain in which Carboxyesterase is Incorporated intoUL39-inactivated HSV

A human carboxyesterase gene having a promoter for the immediate earlygene of human cytomegalovirus was inserted to a lacZ gene (open readingframe) so that DNA in which the lacZ gene was interrupted by the humancarboxyesterase gene was prepared. Vero cells were transfected with thisDNA and infectious DNA derived from UL39-inactivated virus which haslacZ. Produced viruses were collected. Viruses which produced colorlessplaques in the presence of X-gal were collected, followed by plaquecloning. The result antiviruses were stored as a primary stock. Cellsamples were infected with clones obtained as the primary stock. Thecell samples were subjected to a fluorescent antibody technique usingtagged antibodies. For positive clones, a secondary stock was prepared.The finally obtained clones were confirmed to induce carboxyesteraseactivity in infected cells.

Example 4

Isolation of UL56-inactivated HSV-1

HSV-1 virus in which a region including the UL56 gene is deficient wasisolated from strain HF. This HSV-1, HF-derived clone 10 (MNO10:preserved in the Laboratory of Virology, Research Institute for DiseaseMechanism and Control, Nagoya University School of Medicine) wassubjected to PCR, base sequence determination, and Western blot analysisto confirm that the UL56 gene is deficient.

Example 5

Preparation of Attenuated HSV (HR522) Capable of Forming a GiantPolynuclear Cell

Vero cells (RIKEN Cell Bank, Tsukuba Science City, 305 Ibaraki, Japan)were infected with the UL39-attenuated HSV prepared in Example 1 and theHF-derived clone 10 of HSV-1 isolated in Example 4 in which the regionincluding the UL56 gene is deficient. Thereafter, a clone which wascapable both of producing a blue plaque in the presence of X-gal (markerfor UL39-attenuated HSV) and producing a giant polynuclear cell (markerfor HF MNO10) was selected. For confirmation, such a clone was separatedby means of phenotype thereof. Those expressing LacZ (i.e., RR-), andhaving a feature of producing multinucleated giant cell in the infectedcells. The clone obtained from this mixed infection is designated asHR522.

Example 6

Treatment of Cancer Using Attenuated HSV

SW1990 derived from human ovarian cancer was transplanted into groups ofnude mice (female, 6 weeks old, each group includes 10 mice) byintraperitoneally injecting 1×10⁷ cells/mouse in the vicinity of thepancreas under ether anesthesia. After the transplantation, 1×10⁷PFU/mouse of the UL39-inactivated HSV or HR522 was intraperitoneallyinjected into two groups on day 7 and day 12. After the inoculation ofthe UL39-inactivated HSV or HR522, ganciclovir of 0.4 mg/mouse wasadministered to one of each of the UL39-inactivated HSV and HR522inoculation groups on day 10 (i.e., day 17 and 22 after thetransplantation). The survival rate of each group was observed until day60 after the transplantation. The results are shown in FIG. 1.

As can be seen from the result of FIG. 1, it was revealed that thesurvival time of nude mice is elongated by the inoculation of theUL39-inactivated HSV or HR522. Further, it was also revealed that theadministration of ganciclovir further elongates the survival time.Particularly, the group given a combination of the HR522 inoculation andthe ganciclovir administration had a high survival rate.

Example 7

Comparison of Attenuated HSV with Known Anticancer Agents

SW1990 derived from human ovarian cancer was transplanted into groups ofnude mice (female, 6 weeks old, each group includes 10 mice) byintraperitoneally injecting 1×10⁷ cells/mouse in the vicinity of thepancreas under ether anesthesia. After the transplantation, 5×10⁷PFU/mouse of the UL39-inactivated HSV or HR522, or taxol (knownanticancer agent) of 0.4 mg/mouse was intraperitoneally injected intothe respective group on day 7 and day 12. The survival rate of eachgroup was observed until day 60 after the transplantation. The resultsare shown in FIG. 2.

As can be seen from the results shown in FIG. 2, it was revealed thatthe groups to which the UL39-inactivated HSV or HR522 was inoculatedhave a longer survival time than that of the group to which taxol wasadministered.

Example 8

Preparation of US3-deficient Virus

(Mice and Virus)

Female BALB/c mice, 8 to 10 weeks old at the start of treatment, werepurchased from Japan SLC (Hamamatsu, Japan). All of the mice were cagedand maintained in accordance with institutionally recommendedguidelines. A gene recombinant of HSV-2 strain 186 which does not haveUS3 (hereinafter referred to as L1BR1) was prepared by a methoddescribed in “Daikoku, T., Yamashita, Y., Tsurumi, T., Maeno, K., andNishiyama, Y. (1993), Virology 197, 685-694”. As a comparative example,wild-type HSV-2 strain 186 (hereinafter simply referred to as wild type186) and strain YY2 which is a US2-deficient mutant were used. Thesethree viruses were grown in Vero cells and then stored at −80° C. untiluse. Viral infection titers were measured using the Vero cells and wererepresented as plaque forming unit (PFU)/ml.

(HSV Infection Method)

Mice were inoculated intravaginally with the virses by a methoddescribed in “Parr, M. B., Kepple, L., McDermott, M. R., Drew, M. D.,Bozzola, J. J., and Parr, E. R. (1994), Lab. Invest. 70,369-380”, and“Milligan, G. N., and Bernstein, D. I. (1997), Virol. 229, 259-268”. Tosynchronize the estrous cycle at the progesterone-dominated stage priorto viral inoculation, the mice were injected subcutaneously with 0.1μg/mouse of β-estradiol 17-cypionate (hereinafter abbreviated as E)(Sigma, St. Louis, Mo.) with a treatment of 2 mg/mouse of Depo-Provera(hereinafter abbreviated as DP) (Sigma). Five days after theadministration of DP, the mice were swabbed with gauze soaked in sodiumpentbarbital to anesthetize the mice. The mice were intravaginallyinfected with 20 μl of a suspension of wild type 186, wild type YY2, orL1BR1.

(Viral Titration)

PBS (phosphate buffered saline) was placed on the vagina using a pipetteto cause vaginal ravage. Thereafter, the vagina was stored at −80° C. inaccordance with a method described in “Milligan, G. N., and Bernstein,D. I. (1997), Virol. 229, 259-268”, until measurement of infection titerby plaque formation on a monomolecular layer of Vero cells.

(Preparation of Cells in Vagina)

Mononuclear cells (MNC) and epithelial cells (EC) derived from thevagina were obtained in accordance with a method described in“Inagaki-Ohara, K., Nishimura, H., Sakai, T., Lynch, D. H., andyoshikai,Y. (1997), Lab. Invest. 77, 421-429” which is modified from “Rakasz, E.,Hagen M., Sandor, M., and Lynch, R.G. (1997), Int. immunol.9, 161-167”.Briefly, the mice were lethally anesthetized and thereafter the vaginaswere-excised from the mice. The vaginas were cut into 2 mm-sizesections, followed by incubation in Hank's balanced salt solutioncontaining 4 U/ml DNase I at 37° C. for 60 minutes. After shaking, thecell suspensions were passed through nylon wool columns to removenecrotized tissue sections, so that suspensions of single cells wereobtained. The passed cells were centrifuged through a 40%/75% or 25%/40%discontinuous PercolI (Pharmacia, Uppsala, Sweden) gradient at acentrifugal force of 600×g at 20° C. for 20 minutes. MNC and EC werecollected from the interfaces of the 40%/75% and 25%/40%, respectively.Cells isolated from five mice at each day during the time-coursefollowing HSV infection were pooled for the following experiments.

(Flow Cytometry)

Cells were stained by a method described in “Inagaki-Ohara, K.,Kobayashi, N., Nishimura, H., Sakai, T., Matsumoto, Y., Hiromatsu, K.,Awaya, A., and Yoshikai, Y. (1996), Cell immunol. 171, 30-40”.Monoclonal antibodies (mAbs) used in the present invention werepurchased from PharMingen (SanDiego, Calif.): FITC-conjugated anti-CD3mAb (145-2 C11), anti-CD11b mAb (M1/70), anti-CD40 mAb (3/23), anti-IadmAb (AMS-32.1), PE-conjugatedanti-CD4mAb(RM4-5), anti-CD45/B220(RA3-6B2), anti-CD40L mAb (MR1), biotin-conjugated anti-CD8α mAb(53-6.7). Stained cells were analyzed on an EPICS XL flow cytometry(COULTAR, Miami, Fla.).

(Histological Analysis)

Mice were lethally anesthetized and then fixed in PBS (phosphate buffersaline) which contains 4% formaldehyde. The vaginal wall, uterus andrectum were sagittaly cut into blocks and embedded in paraffin.Thereafter, paraffin sections cut 3 μm in thickness were histologicallyexamined by staining with heamatoxylin-eosin. Also, the paraffinsections were immunohistochemically analyzed using a rabbit anti-HSV-2antiserum in accordance with a method described in “Kurachi, R.,Daikoku, T., Tsurumi, T., Maeno, K., Nishiyama, Y., Kurata, T. (1993),Arch Virol. 133, 259-273”. Briefly, the sections were deparaffinized andthen treated with PBS containing 0.02% (v/v) calcium chloride and 0.25%(v/v) trypsin (Difco, Detroit, Mich.) at 37° C. for 30 minutes, and thenimmersed in 0.3 volume % hydrogen peroxide methanol solution for 30minutes. The sections were reacted with anti-HSV-2 antiserum at 4° C.overnight. Biotinylated anti-rabbit IgG (DAKO Japan, Tokyo, Japan) wasadded to the sections, and allowed to react at 37° C. for 30 minutes.Thereafter, peroxidase-conjugated streptoavidin (DAKO Japan) was addedand then allowed to react at 37° C. for 30 minutes. The peroxidasereaction was conducted in 0.05 M Tris buffer (pH 7.6) containing 0.02%diaminobenzidine (Chemical Dojin Inc., Kumamoto, Japan) and 0.015%hydrogen peroxide. Cell nuclei were counter-stained with 2% methylgreen(Chrome, Stuttgart, Germany).

(Measurement of Cytokine in Vaginal Washes by ELISA)

Vaginal washes were collected daily after infection. Cytokine content inthe vagina washes was analyzed using mouse IFN-γ, IL-4 (BioSourceInternational, Inc., Camarillo, Canada) and IL-12 with a measurement kitusing ELISA (Amersham LIFE SCIENCE, Buckinghamshire, Britain) inaccordance with the manufacturer's instructions.

(Statistical Analysis)

The t-test was used to determine significant differences. A p value ofless than 0.05 is considered significant.

(Lesions of Genital Organs and Mortality Induced by Wild Type 186, YY2(US2-deficient Strain 186) or L1BR1 (US3-deficient Strain 186))

To determine the effects of US2 and US3 deletion on pathogenicity, micewere intravaginally infected with 2×10⁴ PFU of YY2or L1BR1. FIG. 3 showsthe representative appearance of the mice after the vaginal infection.The genital organ infected with YY2 started swelling significantly onday 5 after the inoculation (post inoculation; p.i.) as well as the miceinfected with wild type 186. For the mice infected with L1BR1,substantially no symptom of vaginal inflammation was observed. Thesurvival rate was shown in FIG. 4. All of the mice infected with wildtype 186 or YY2 died within 10 days after the intravaginal inoculation.In contrast, all of the mice infected with L1BR1 survived as long asthree weeks after the inoculation.

(Clearance of Virus in the Vagina)

To compare the rates of viral clearance in vaginal mucosa, mice wereintravaginally infected with wild type 186, YY2, or L1BR1 and thereafterviral titers were measured (FIG. 5). The viral titers of the micerapidly increased and reached the maximum levels at one daypost-infection (p.i.). The titers of the mices infected with YY2 andL1BR1 gradually decreased until day 5, while the titers of the miceinfected with L1BR1 sharply decreased after day 5. The titers of themice infected with wild type 186 were sustained until day 3 andthereafter decreased slowly.

(Histopathological Changes of the Vagina)

The pathological changes of the vagina and uterus in mice sacrificed at1, 3, 5 and 7 days post-infection (p.i.) were examined. For all of themice, viral infection was observed in squamous cells in the epithelialtissue on day 1 (FIGS. 6A, 6B and 6C). By histological analysis,multinucleated giant cells and intranuclear inclusions were observed insquamous cells, and HSV-2 antigens were also detected in these cells(FIGS. 6D, 6E and 6F). Until day 3, no significant changes were observedin the lesions. In this example, viral infection extended to thesquamous cells in the epithelial tissue of the vulva and the skin, andreached the nerve plexus in the subepithelium of the mice infected withwild type 186 and YY2. However, such viral spread was not observed inthe mice infected with L1BR1. Instead, sub-epithelial infiltration ofmononuclear cells was observed on day 5 post inoculation (p.i.) (FIGS.6G, 6H and 6I).

(Reaction of Cells in the Vagina)

Next, the number of mononuclear cells (MNC) isolated from the vagina wasmeasured. As shown in FIG. 7A, the number of MNC in the mice infectedwith L1BR1 increased until day 5 and thereafter rapidly decreased.However, for the mice infected with wild type 186 or YY2, the numbers ofMNC increased on day 1 but thereafter did not increase. On day 3 postinoculation (p.i.), a slow decrease was observed. Such specific reactionkinetics is closely related to histological changes in the vaginal wall.As can be seen from these results, a delay in clearing viruses in themice infected with wild type 186 or YY2 led to a high case fatalityrate.

(Measurement of Expression Level of Fas Antigen)

As shown in FIG. 7B, the number of Fas⁺ cells was greater in the miceinfected with L1BR1 than the mice infected with wild type 186 or YY2until day 5. Most of the Fas⁺ cells were positive for MHC class IIantigen I-A^(d) (data not shown). According to this, it was found thatthe number of Fas⁺ vaginal ECs was significantly greater in the miceinfected with L1BR1 than in the mice infected with wild type 186 or YY2at the early phase of the infection.

(Measurement of Reaction Kinetics of Appearance of APC and T Cells inVagina)

As can also be seen from FIG. 8, CD40 and CD11b were used asrepresentative markers for DC and Mφ, respectively. The number of CD40⁺cells rapidly increased in the mice infected with L1BR1, and wasconsistently greater. than in the mice infected with wild type 186 orYY2 throughout the experiment. On day 3, the number of CD11b⁺ cells onlyslightly increased. However, on day 5, a significant increase wasobserved in the mice infected with L1BR1. Concerning T cells, thenumbers of CD4⁺, CD8⁺, CD40L⁺ and Fas⁺ T cells reached the respectivemaximum levels on day 5 and thereafter decreased. CD40L as well as Faswas expressed in activated T cells. The numbers of Fas⁺ and CD40L⁺ cellswas much greater in the mice infected with L1BR1 than in the miceinfected with wild type 186 or YY2. The numbers reached the respectivemaximum levels on day 5. These results indicate thatinduction/activation of vaginal APC and T cells in the mice infectedwith L1BR1 is more rapid and greater in magnitude than in the miceinfected with wild type 186 or YY2.

(Cytokines Secretion in Vaginal Washes)

Production of cytokines is required for protecting hosts from viralpathogens. The production level of cytokines was examined on day 2 byELISA. As a result, an increase in production of IL-12 was detected. Onday 2 and 4, the IL-12 production was significantly greater in the miceinfected with L1BR1 than in the mice infected with wild type 186 or YY2(FIG. 9). IFN-γ and IL-4 are representative cytokines of Th1 and Th2,respectively. The production pattern of IFN-γ was significantly higherin the mice infected with L1BR1 than in the mice infected with wild typestrain 186 on day 4. The production level of IL-4 tended to be lower inthe mice infected with L1BR1 than in the mice infected with wild typestrain 186. The production patterns and levels of both cytokines in themice infected with YY2 were similar to those in the mice infected withwild type 186. These results indicate that cytokine production biasedtowards Th1 was induced in the mice infected with L1BR1.

The US3-deficient HSV gene recombinant contained in the vaccines of thepresent invention was considerably attenuated. Therefore, when theUS3-deficient HSV gene recombinant is inoculated into an organism, theorganism exhibits no or substantially no symptom of the infection.Nevertheless, the US3-deficient HSV gene recombinant can activate theimmune function of infected patients against HSV infection. Therefore,the severity and/or infection rate of HSV infection can be reduced, sothat diseases caused by HSV infection can be prevented and/or treated.Since the US3 gene is not necessarily essential for virus replication,the US3-deficient HSV gene recombinant can be multiplied within the bodyof an organism to which the gene recombinant has been inoculated, sothat immune activity against HSV infection can be sustained for a longtime.

Example 9

Comparison of HF (MNO10) with Antitumor Function of UL39-inactivated HSV

Tumor cell strain NfSaY83 derived from C3H mouse was transplanted intogroups of CH3 mice (6 weeks old, each group includes 10 mice) byintraperitoneally injecting 1×10⁷ cells/mouse under ether anesthesia.After the transplantation, 1×10⁷ PFU/mouse of UL39-inactivated virus orHF strain clone 10 (UL56-deficient virus) was intraperitoneally injectedinto the respective groups on day 7. Second injection was conducted forthe group, to which the UL39-inactivated virus had been administered, onday 12 after the transplantation. The survival time was compared. Onlycancer cells were inoculated to mice as a control. The results are shownin FIG. 11.

The results of FIG. 11 clearly indicate that all of the groups exhibiteda survival rate of 100% until day 13, thereafter the survival time wasmore elongated when HF strain clone 10 was inoculated once than when theUL39-inactivated virus was inoculated twice.

Example 10

Effect of Consecutive Administration of HF (MNO10)

Tumor cell strain NfSaY83 derived from C3H mouse was transplanted intogroups of CH3 mice (6 weeks old, each group includes 10 mice) byintraperitoneally injecting 5×10⁶ cells/mouse under ether anesthesia.After the transplantation, 1×10⁷ PFU/mouse of HF (UL56-deficient virus)was intraperitoneally injected into one group on three consecutive days,i.e., day 7, 8 and 9. The survival time was compared. Only cancer cellswere inoculated to mice as a control. The results are shown in FIG. 12.

The results of FIG. 12 indicate that all of the groups exhibited asurvival rate of 100% until day 17 and thereafter, the survival time wasmore elongated by consecutively administering the virus.

Example 11

1×10⁷ PFU of the attenuated HSV of the present invention (MNO10), 1.8 gof glucose, and 0.06 g of sodium citrate were dissolved in 80 ml ofwater for injection. The solution was adjusted to pH 6.3 by addingsodium hydrate, and to a total volume of 100 ml by adding water forinjection. Thereafter, the resultant solution was subjected tofiltration, and then 500 ml thereof was loaded into a gas-permeableplastic container (made of PP). This container was subjected to steamsterilization under high pressure at 116° C. for 14 minutes. Thereafter,the container was wrapped with a secondary wrapping material (aluminadeposition film), followed by vacuum packaging or nitrogen gas filling,thereby obtaining an injection.

When the attenuated HSV of the present invention in which UL56 isinactivated is used, a single administration can prolong the life timemore than when the UL39-inactivated virus, i.e., attenuated HSV strainis consecutively administered.

Further, if the attenuated HSV of the present invention is consecutivelyadministered, a higher level of life prolongation effect can beobtained. Therefore, it was demonstrated that the attenuated HSV of thepresent invention is useful for pharmaceutical compositions for treatingmalignant tumor.

Example 12

Antitumor Action of US3-deficient HSV-2 Virus and US3-andUL56-inactivated HSV (HL)

Next, the US3-deficient HSV-2 virus and the US and UL56-inactivated HSV(HL) were examined for their antitumor functions.

(Viruses Used)

The following viruses were used in Example 9. The structure of theviruses is shown in FIG. 13.

1) HSV-2 US3Δ (L1BR1)

Mutated virus derived from wild type HSV-2 (strain 186), in which theUS3. gene was inactivated by inserting a lacZ cassette. This virus wasprepared as described in Example 4.

2) HSV HL

Recombinant virus from HSV-1 HF and HSV-2 US3Δ. The L region thereof ismainly derived from HSV-1 HF, and the S region thereof is derived fromHSV-2. This recombinant virus is prepared by mixed infection with theabove-described two viruses, forming cell fusion, and separating virusesforming a blue plaque in the presence of X-gal. Thereafter, thestructure of the gene was confirmed by PCR. HL virus lacks the US3 andUL56 genes.

3) HSV-1 UL39-inactivated HSV

The viruses used in the above-described examples were used as controls.

(Mice)

Tumor cell strain NfSaK derived from C3H mouse was transplanted intogroups of CH3 mice (6 weeks old, each group includes 10 mice) byintraperitoneally injecting 1×10⁷ cells/mouse at their backs and necksunder ether anesthesia. On day 7 after the transplantation, 1×10⁷PFU/mouse of HSV-2 US3Δ, or PBS (control), was injected into the mice inwhich tumor cells were injected to the backs thereof, while 1×10⁷PFU/mouse of HL and HrR3, or PBS (control), were injected into the micein which tumor cells were injected to the necks thereof. The survivaltime was compared. The results are shown in the following Table andFIGS. 14 and 15.

TABLE Action of HSV-2 US3Δ on melanoma solid tumor (back) (Number ofsurviving mice (rate)) Control (PBS inoculation) Day 1–16 5/5 (100%) Day17 4/5 Day 24 2/5 Day 25 1/5 Day 37 0/5 (0%) HSV-2 US3Δ inoculated groupDay 1–26 5/5 (100%) Day 27 4/5 Day 32 3/5 Day 42 2/5 Day 60 2/5 (40%)

Action of HSV on NfSak solid tumor (neck) (Number of surviving mice(rate)) Control (PBS inoculation) Day 1–22 5/5 (100%) Day 23 4/5 Day 253/5 Day 30 2/5 Day 31 0/5 (0%) HL inoculated group Day 1–27 7/7 (100%)Day 28 6/7 Day 30 5/7 Day 32 4/7 Day 60 4/7 (57%) HSV-1 UL39-inactivatedHSV inoculated group Day 1–23 7/7 (100%) Day 24 6/7 Day 25 5/7 Day 344/7 Day 35 3/7 Day 36 2/7 Day 60 2/7 (28%)

It was demonstrated that when the attenuated HSV of the presentinvention in which US3 is inactivated is used, a single administrationcan prolong the life time.

Example 13

Antitumor Effect of Hh

Next, the antitumor effect of the UL39- and UL56-inactivated HSV (Hh)was demonstrated.

BALB/c mice (6 weeks old, each group includes 6 to 7 mice) were used assubject animals. Cancer Colon26 (Dr. Shuji Hayashi, Department ofSurgery, Nagoya University School of Medicine; a cell strain derivedfrom large intestine cancer of BALB/c mice) was intraperitoneallyinoculated at 5×10⁶cells/mouse. HSV-1 Hh prepared in Example 2 was usedas the virus control in PBS inoculation.

(Mode 1)

After the inoculation of the tumor cells, 1×10⁷ PFU of Hh virus wasintraperitoneally inoculated on day 7 and its survival curve wascompared with the controls (PBS inoculation). The results are shownbelow.

TABLE Antitumor effect of Hh Days after antitumor inoculation Day 15 Day30 Day 45 Day 60 Day 75 Hh 6/6 5/6 4/6 4/6 4/6 inoculation group Control6/6 5/6 4/6 2/6 2/6 group

(Mode 2)

After the inoculation of tumor cells, 1×10⁷ PFU of Hh virus wasinoculated consecutively on day 8, 9 and 10 and its survival curve wascompared with the controls. The results are shown below.

TABLE 2 Effect of consecutive three administrations Days after antitumorinoculation Day 30 Day 45 Day 60 Hh 7/7 7/7 7/7 inoculation groupControl group 5/7 4/7 2/7

As described above, Hh exhibited a significant antitumor effect ascompared to the control group. Particularly, Hh having a significantlyimproved safety led to 100% survival when administered three consecutivetimes. Moreover, when these surviving mice were challenged again withColon26 at 1×10⁷ cells/mouse, 100% of the mice survived. Thisdemonstrated establishment of antitumor immunity.

Example 14

L1BR1

In this example, the effect of the gene recombinant HSV-2186, which doesnot express US3, as prepared in Example 9 on solid tumor wasdemonstrated.

Female mice C3H/He (6 weeks; each group includes 5 mice) were used assubject animals. As tumor cells, NfSaY83 used in the above-describedexamples was used.

The hair of the back was shaved and 1.25×10⁶ cells/0.1 ml of NfSa wasinoculated on the back.

After the inoculation of the tumor cells, 0.1 ml of L1BR1 (1×10⁷ cellPFU/0.1 ml) was inoculated to a tumor portion three times.

Evaluation was carried out based on the number of surviving mice andchange in the size of tumor (volume). The results are shown in thefollowing table.

TABLE Transition in volume (mm³) of tumor after inoculation of tumor(individual results) Day 10 Day 20 Day 30 Day 40 Day 50 L1BR1 5 <5 <5 <5<5 inoculation 9 54 150 545 Dead group <5 <5 <5 <5 <5 24 <5 <5 <5 <5 13<5 <5 <5 <5 Number of 5/5 5/5 5/5 5/5 4/5 surviving mice Control 21 1861210 Dead group 42 419 Dead 73 420 Dead 42 161 1346 4300 Dead 142 266486 Dead Number of 5/5 5/5 3/5 1/5 0/5 surviving mice

As described above, L1BR1 (US-deficient HSV-2) exhibited an excellentantitumor effect on solid tumor.

Example 15

Use of Attenuated HSV for HIV Therapy

Next, it is demonstrated that another disease can be treated orprevented using the attenuated HSV of the present invention.

A nucleic acid sequence having a promoter for the immediate early geneof human cytomegalovirus at its upstream portion and a sequence encodingan envelope protein, HIV gp120, is inserted to a lacZ gene (open readingframe) so that DNA in which the lacZ gene is interrupted by the necleicacid sequence is prepared.

Vero cells are transfected with this DNA and infectious DNA derived fromthe UL56- and UL39-inactivated virus (the attenuated HSV (HR522)prepared in Example 5). Produced viruses are collected.

Viruses which produced colorless plaque in the presence of X-gal arecollected, followed by plaque cloning. The resultant viruses are storedas a primary stock. Cell samples are infected with clones obtained asthe primary stock. The infected cell samples are subjected to afluorescent antibody technique using tag antibodies. For positiveclones, a secondary stock is prepared. The finally obtained clones aresubjected to dot blotting using commercially available anti-gp120antibodies to test the presence of gp120. A gp120-positive strain isused in the following experiment.

Thereafter, the attenuated HSV which expressed HIV gp120 can be used toprevent a human from being infected with HIV or treat a human infectedwith HIV. The effect of the attenuated HSV of the present invention maybe advantageous over conventional drug therapies.

As the above-described HIV antigen, gp41 and gp17 matrix proteins, p24capsid protein, reverse transcriptase (HIV pol), tat rev, and the likemay be used as well as gp120.

Therefore, it is demonstrated that the attenuated HSV of the presentinvention is effective to an infectious disease caused by an infectouspathogen other than herpes virus-related pathogens.

INDUSTRIAL APPLICABILITY

The present invention provides a method for effectively treating variousdiseases (e.g., infectious diseases or cancer). The present inventionprovides a sustaining effect, with few side effects, against herpesvirus-related diseases, tumor, and the like, for which there isconventionally no effective treatment method. These are considered asadvantageous effects. Production of compositions, virus constructs,kits, medicaments, and the like for use in the treatment method of thepresent invention is sufficiently industrially applicable. The presentinvention (compositions and the like) can be practiced by doctors andfurther, for example, pharmaceutical companies in their business.Therefore, the present invention is considered to be sufficientlyindustrially applicable. The method of the present invention is usefulfor a therapeutic method for purely therapy purposes and furtherclinical trial for business purposes. Therefore, the method of thepresent invention is industrially applicable. The therapeutic method ofthe present invention may be practiced indirectly or directly in themarginal industries of medical business, and therefore is sufficientlyindustrially applicable.

The invention claimed is:
 1. A herpes virus, wherein at least twonon-essential genes for replication thereof are inactivated and furtherwherein the herpes virus is herpes simplex virus, wherein the firstnon-essential gene is UL56 and the second non-essential gene forreplication is UL43.
 2. The herpes virus according to claim 1, whereinthe second non-essential gene for replication is a gene not involved inDNA and deoxyribonucleotide metabolism.
 3. The herpes virus according toclaim 2, wherein the gene not involved in DNA and deoxyribonucleotidemetabolism is UL43.
 4. The herpes virus according to claim 1, whereinthe second non-essential gene for replication is UL43.
 5. The herpesvirus according to claim 1, further comprising an exogenous suicidegene.
 6. The herpes virus according to claim 1, further comprising acarboxyesterase gene.
 7. The herpes virus according to claim 1, whereinthe virus has ability to select cancer cells.
 8. The herpes virusaccording to claim 1, wherein the inactivation includes at least onenucleotide substitution, addition, deletion, or modification in thesequence of the non-essential gene for replication.
 9. The herpes virusaccording to claim 1, wherein the virus is a modified HSV-1 or HSV-2.